Process for enzymatically modifying pectin

ABSTRACT

A process for treating a pectin with a pectin methyl esterase (PME) is descibed. Here, the PME is not a plant PME—but the PME is capable of exhibiting at least one plant PME property; wherein the at least one plant PME property comprises at least block—wise de-esterification of the pectin.

The present invention relates to a process.

In particular, the present invention relates to a process whichcomprises the use of an enzyme.

More in particular, the present invention relates to a process forenzymatically modifying pectin.

Pectin is a structural polysaccharide commonly found in the form ofprotopectin in plant cell walls. The backbone of pectin comprises linked(1→4)-α-D-galacturonic acid residues which are interrupted with a smallnumber of 1→2 linked α-L-rhamnose units.

In addition, pectin comprises highly branched regions with an almostalternating rhatnno-galacturonan chain. These highly branched regionsalso contain other sugar units (such as D-galactose, L-arabinose andxylose) attached by glycosidic linkages to the C3 or C4 atoms of therhamnose units or the C2 or C3 atoms of the galacturonic acid units. .The long chains of -(1→4)-αlinked galacturonic acid residues arecommonly referred to as “smooth” regions, whereas the highly branchedregions are commonly referred to as the “hairy regions”.

Some of the carboxyl groups of the galacturonic residues are esterified(e.g. the carboxyl groups are metbylated). By way of example somegalacturonic acid residues are esterified with methanol. Typicallyesterification of the carboxyl groups occurs after polymerisation of thegalacturonic acid residues. However, it is extremely rare for all of thecarboxyl groups to be esterified (e.g. methylated).

Usually, the degree of esterification will vary from 0-90%. If 50% ormore of the carboxyl groups are esterified then the resultant pectin isreferred to as a “high ester pectin” (“HE pectin” for short) or a “highmethoxyl pectin”. If less than 50% of the carboxyl groups are esterifiedthen the resultant pectin is referred to as a “low ester pectin” (“LEpectin” for short) or a “low methoxyl pectin”. If 50% of the carboxylgroups are esterified then the resultant pectin is referred to as a“medium ester pectin” (“ME pectin” for short) or a “medium methoxylpectin”. If the pectin does not contain any—or only a few—esterifiedgroups it is usually referred to as pectic acid.

The structure of the pectin, in particular the degree of esterification(e.g. methylation), dictates many of the resultant physical and/orchemical properties of the pectin. For example, pectin gelationdepends.on the chemical structure of the pectin, especially the degreeof esterification. In addition, however, pectin gelation also depends onthe soluble-solids content, the pH and calcium ion concentration. Withrespect to the latter, it is known that the calcium ions form complexeswith free carboxyl groups, particularly those on a LE pectin.

Pectic enzymes are classified according to their mode of attack on thegalacturonan part of the pectin molecule. A review of some pecticenzymes has been prepared by Pilnik and Voragen (Food Enzymology, Ed.:P. F. Fox; Elsevier; (1991); pp: 303-337). In particular, pectinmethylesterases (EC 3.1.1.11), otherwise referred to as PMEs,de-esterify HE pectins to LE pectins or pectic acids. In contrast, andby way of example, pectin depolymerases split the glycosidic linkagesbetween galacturonosyl methylester residues.

In more detail, PME activity produces free carboxyl groups and freemethanol. The increase in free carboxyl groups can be easily monitoredby automatic titration. In this regard, earlier studies have shown thatsome PMEs de-esterify pectins in a random manner, in the sense that theyde-esterify any of the esterified (e.g. methylated) galacturonic acidresidues on one or more than one of the pectin chains. Examples of PMEsthat randomly de-esterify pectins may be obtained from fungal sourcessuch as Aspergillus aculeatus (see WO 94/25575) and Aspergillusjaponicus (Ishii et al 1980 J Food Sci 44 pp 611-14). Baron et al (1980Lebensm. Wiss. μ-Technol. 13pp 330-333) apparently have isolated afungal PME from Aspergillus niger. This fungal PME is reported to have amolecular weight of 39000 D, an isoelectric point of 3.9, an optimum pHof 4.5 and a K_(m) value (mg/ml) of 3.

In contrast, some PMEs are known to de-esterify pectins in a block-wisemanner, in the sense that it is believed they attack pectins either atnon-reducing ends or next to free carboxyl groups and then proceed alongthe pectin molecules by a single-chain mechanism, thereby creatingblocks of un-esterified galacturonic acid units which can be calciumsensitive. Examples of such enzymes that block-wise enzymaticallyde-esterify pectin are plant PMEs. Up to 12 isoforms of PME have beensuggested to exist in citrus (Pilnik W. and Voragen A. G. J. (FoodEnzymology (Ed.: P. F. Fox); Elsevier; (1991); pp: 303-337). Theseisoforms have different properties.

Random or blockwise distribution of free carboxyl groups can bedistinguished by high performance ion exchange chromatography (Schols etal Food Hydrocolloids 1989 6 pp 115-121). These tests are often used tocheck for undesirable, residual PME activity, in citrus juices afterpasteurisation because residual PME can cause, what is called, “cloudloss” in orange juice in addition to a build up of methanol in thejuice.

PME substrates, such as pectins obtained from natural plant sources, aregenerally in the form of a high ester pectin having a DE of about 70%.Sugar must be added to extracts containing these high ester PMEsubstrates to provide sufficient soluble solids to induce gelling.Usually a minimum of 55% soluble solids is required.

Syneresis may occur. By way of example, syneresis in marmalades and jamswith low soluble solid content (<55%) may occur when using HE-pectin.However, HE-pectin is not usually used in such applications. If pectinsare to be used, then typically amidated pectins or LE-pectin are used,such as for jams with <55% SS.

When syneresis does occur, expensive additives must be used to inducegelline.

Versteeg et al (J Food Sci 45 (1980) pp 969-971) apparently haveisolated a PME from orange. This plant PME is reported to occur inmultiple isoforms of differing properties. Isoform I has a molecularweight of 36000 D, an isoelectric point of 10.0, an optimum pH of 7.6and a K_(m) value (mg/ml) of 0.083. Isoform II has a molecular weight of36200 D. an isoelectric point of 11.0, an optimum pH of 8.8 and a K_(m)value (mg/ml) of 0.0046. Isoform III (HMW-PE) has a molecular weight of54000 D, an isoelectric point of 10.2, an optimum pH of 8 and a k_(m)value (mg/ml) of 0.041. However, to date there has been very limitedsequence data for such PMEs.

According to Pilnik and Voragen (ibid), PMEs may be found in a number ofother higher plants, such as apple, apricot, avocado, banana, berries,lime, grapefruit, mandarin, cherries, currants, grapes, mango, papaya,passion fruit, peach, pear, plums, beans, carrots, cauliflower,cucumber, leek, onions, pea, potato, radish and tomato. However,likewise, so far there has been very limited sequence data for suchPMEs.

A plant PME has been reported in WO-A-97/03574 (the contents of whichare incorporated herein by reference). This PME has the followingcharacteristics: a molecular weight of from about 36 kD to about 64 kD;a pH optimum of pH 7-8 when measured with 0.5% lime pectin in 0.15 MNaCl; a temperature optimum of at least 50° C.; a temperature stabilityin the range of from 10°—at least 40° C.; a k_(m) value of 0.07%; anactivity maximum at levels of about 0.25 M NaCl; an activity maximum atlevels of about 0.2 M Na₂SO₄; and an activity maximum at levels of about0.3 M NaNO₃.

Another PME has been reported in WO 97/31102 (the contents of which areincorporated herein by reference).

PMEs have important uses in industry. For example, they can be used inor as sequestering agents for calcium ions. In this regard, andaccording to Pilnik and Voragen (ibid), cattle feed can be prepared byadding a slurry of calcium hydroxide to citrus peels after juiceextraction. After the addition, the high pH and the calcium ionsactivate any native PME in the peel causing rapid de-esterification ofthe pectin and calcium pectate coagulation occurs. Bound liquid phase isreleased and is easily pressed out so that only a fraction of theoriginal water content needs to be removed by expensive thermal drying.The press liquor is then used as animal feed.

As indicated above, a PME has been obtained from Aspergillus aculeatus(WO 94/25575). Apparently, this PME can be used to improve the firmnessof a pectin-containing material, or to de-methylate pectin, or toincrease the viscosity of a pectin-containing material.

It has also become common to use PME in the preparation of foodstuffsprepared from fruit or vegetable materials containing pectin—such asjams or preservatives. For example, WO-A-94/25575 further reports on thepreparation of orange marmalade and tomato paste using PME obtained fromAspergillus aculeatus.

JP-A-63/209553 discloses gels which are obtained by the action of pectinmethylesterase, in the presence of a polyvalent metal ion, on a pecticpolysaccharide containing as the main component a high-methoxyl polyα-1,4-D-galacturonide chain and a process for their production.

Pilnik and Voragen (ibid) list uses of endogenous PMEs which includetheir addition to fruit juices to reduce the viscosity of the juice ifit contains too much pectin derived from the fruit, their addition aspectinase solutions to the gas bubbles in the albedo of citrus fruitthat has been heated to a core temperature of 20° C. to 40° C. in orderto facilitate removal of peel and other membrane from intact juicesegments (U.S. Pat. No. 4,284,651), and their use in protecting andimproving the texture and firmness of several processed fruits andvegetables such as apple (Wiley & Lee 1970 Food Technol 24 1168-70)canned tomatoes (Hsu et al 1965 J Food Sci 30 pp 583-588) and potatoes(Bartolome & Hoff 1972 J Agric Food Chem 20 pp 262-266).

Glahn and Rolin (1994 Food Ingredients Europe, Conf Proceedings pp252-256) report on the hypothetical application of the industrial “GENUpectin type YM-100” for interacting with sour milk beverages. No detailsare presented at all on how GENU pectin type YM-100 is prepared.

EP-A-0664300 discloses a chemical fractionation method for preparingcalcium sensitive pectin. This calcium sensitive pectin is said to beadvantageous for the food industry.

Plastow G. S. (1988 Molecular Microbiology 2(2) 247-254) reports on apectin methyl esterase gene of Erwinia chrysanthemi B374. According tothe author:

“The isolation of the Erwinia gene provides a simple method for theproduction of PME free from depolymerizing pectinases thereby extendingits potential uses”.

Plastow G. S. further states that:

“The PME . . . produced from E.coli supernatants has been usedsuccessfully to de-esterify high-methoxyl pectin for the production ofcalcium-set gels. The gels that were obtained were found to equal thosemade from pectate produced using enzyme extracted from orange peel ( . .. unpublished results).”

Thus, pectins and de-esterified pectins, in addition to PMEs, have anindustrial importance.

However, and as reported in PCT/IB98/00673 (filed Apr. 24, 1998), abenefit derived from use of a PME in the preparation of, for example, afoodstuff will depend to some extent on the quality and quantity andtype of the PME used and on the qualiy and quantity and type of the PMEsubstrates—in particular pectin—that may be present in the material usedto prepare the foodstuff. For example, if the substrate is a fruitmaterial or a vegetable material then the amount and/or structure ofnatural pectin in that substrate will be different for different typesof fruit material or vegetable material. This is also borne out by thedata presented in WO-A-94/25575, especially FIG. 7 where it is clear tosee that its PME system is not ideal.

According to a first aspect of the present invention there is provided aprocess for treating a pectin with a pectin methyl esterase (PME);wherein the PME is not a plant PME; but wherein the PME is capable ofexhibiting at least one plant PME property; and wherein the at least oneplant PME property comprises at least block-wise de-esterification ofthe pectin.

According to a second aspect of the present invention there is provideda PME treated pectin prepared by the process according to the presentinvention.

According to a third aspect of the present invention there is provided afoodstuff comprising a PME treated pectin prepared by the processaccording to the present invention.

According to a fourth aspect of the present invention there is provideduse of a PME as herein defined to reduce the number of ester groups in apectin and in a block-wise manner.

According to a fifth aspect of the present invention there is providedthe use of a PME as herein defined to de-esterify two or more adjacentgalacturonic acid residues of a pectin on at least substantially all ofthe pectin chains.

The present invention also relates to any one or more of:

a construct expressing or comprising the PME as defined herein or thenucleotide sequence as defined herein.

a vector expressing or comprising a construct of the present inventionor the PME as defined herein or the nucleotide sequence as definedherein.

a combination of constructs comprising at least a first constructexpressing or comprising the PME enzyme as defined herein or thenucleotide sequence as defined herein; and a second construct comprisinga gene of interest (GOI) and a promoter.

a cell, tissue or organ expressing or comprising a vector according tothe present invention or a construct according to the present inventionor the PME as defined herein or the nucleotide sequence as definedherein or the combination of constructs according to the presentinvention.

a transgenic organism expressing or comprising a cell, tissue or organexpressing or comprising a vector according to the present invention ora construct according to the present invention or the PME as definedherein or the nucleotide sequence as defined herein or the combinationof constructs according to the present invention.

a recombinant PME enzyme which is immunologically reactive with anantibody raised against a PME enzyme as defined herein.

In addition to the sequences presented in the attached sequence listings(as well as fragments, derivatives or homologues thereof), the presentinvention also covers sequences that are complementary to theaforementioned sequence listings (as well as fragments, derivatives orhomologues thereof). The present invention also covers sequences thatcan hybridise to the aforementioned sequence listings (as well asfragments, derivatives or homologues thereof). The present inventionalso covers sequences that are complementary to sequences that canhybridise to the aforementioned sequence listings (as well as fragments,derivatives or homologues thereof).

The present invention also relates to novel amino acid sequences andnovel nucleotide sequences presented herein.

Preferably, those novel amino acid sequences and novel nucleotidesequences are isolated and/or purified. Here the term “isolated” and“purified” refer to molecules, either nucleic or amino acid sequences,that are removed from their natural environment and isolated orseparated from at least one other component with which they arenaturally associated.

The present invention is based on the highly surprising finding that itis possible to obtain PMEs from sources other than plants that arecapable of block-wise de-esterifying pectins but wherein those PMEs haveplant PME like properties.

More in particular, the present invention is based on the highlysurprising finding that it is possible to obtain PMEs from bacterialsources that are capable of block-wise de-esterifying pectins.

The present invention is distinguishable over the teachings of, forexample, Plastow G. S. (ibid) as that author does not discloseblock-wise de-esterification of pectins. Moreover, that author refers tounpublished work that includes a comparison with an “enzyme extractedfrom orange peel”—and yet no details are provided on what enzyme, letalone enzymatic activity, is used in the comparative studies. Moreover,the reference to “calcium set” gels and the comparison to pectateproduced gels in that paper indicate that the pectins were de-esterifiedto low ester pectins—which is in direct contrast to a highly preferredaspect of the present invention.

Thus, the present invention relates to a process for treating a pectinwith a PME; wherein the PME is not a plant PME; but wherein the PME iscapable of exhibiting at least one plant PME property; and wherein theat least one plant PME property comprises at least block-wisede-esterification of the pectin.

Preferably, the PME has a molecular weight of about 36.000 D and/or a pIof about >9 and/or a pH optimum with lime pectin (as determined by theaforementioned method) of about pH 7 and/or a temperature optimum withlime pectin (as determined by the aforementioned method) of about 48° C.

Preferably, the PME comprises the amino acid sequence shown as SEQ.I.D.No.2 or a variant, derivative or homologue thereof, includingcombinations thereof.

Preferably, the PME has the amino acid sequence shown as SEQ.I.D. No.2,or a variant, derivative or homologue thereof.

Preferably, the PME has the amino acid sequence shown as SEQ.I.D. No.2.

Preferably, the PME has been expressed by a nucleotide sequencecomprising the nucleotide sequence shown as SEQ.I.D. No. 1, or avariant, derivative or homologue thereof, or combinations thereof.

Preferably, the PME has been expressed by a nucleotide sequence havingthe nucleotide sequence shown as SEQ.I.D. No. 1 or a variant, derivativeor homologue thereof.

Preferably, the PME has been expressed by a nucleotide sequence havingthe nucleotide sequence shown as SEQ.I.D. No. 1.

Preferably, the PME has been prepared by use of recombinant DNAtechniques.

Preferably, the PME is obtainable from a micro-organism, preferably abacterium.

Preferably, the pectin is treated by the PME in the presence of sodiumions.

Preferably, the sodium ions are derived from NaCl, NaNO₃ or Na₂SO₄ orcombinations thereof.

Preferably, the process includes the further step of isolating the PMEtreated pectin from the active PME. Here, the PME treated pectin can bephysically removed from the active PME or vice versa. Preferably,however, the PME treated pectin is isolated from the active PME bysimply inactivating the PME, such as through the application of heat.

Preferably, the PME treated pectin is a high ester pectin.

Preferably, the PME treated pectin contains from about 70% to about 80%ester groups.

Preferably, the PME treated pectin contains from about 72% to about 80%ester groups.

Preferably, the PME treated pectin contains from about 74% to about 80%ester groups.

Preferably, the PME treated pectin contains from about 76% to about 80%ester groups.

Preferably, the PME treated pectin contains from about 77% to about 79%ester groups.

Preferably, the PME treated pectin contains about 78% ester groups.

Preferably, the process includes the further step of adding the PMEtreated pectin to a medium that is suitable for consumption.

Preferably, the medium is an aqueous solution.

Preferably, the medium is an acidic environment.

Preferably, the acidic environment has a pH of from about 3.5 to about5.5, preferably wherein the acidic environment has a pH of from 4 toabout 5.5.

Preferably, the acidic environment has a pH of about 4.

Preferably, the aqueous solution is a beverage.

Preferably, the beverage is an acidified milk drink, drinking yoghurt, amilk drink comprising fruit, or a beverage enriched with proteins, suchas plant and/or dairy proteins, such as whey protein and/or soyaprotein. A protocol for determining the suitability of a treated pectinfor use in a drink is shown after the Examples Section.

Acidified milk drinks with loner shelf life are very popular, especiallyin the Far East. In some cases, a heat treatment is necessary to obtaina long shelf life. In order to avoid sedimentation of protein during andafter heating, pectin is added as a stabilising agent. In someapplications, the quality of the acidified milk drink may depend on theproperties and the concentration of the pectin used.

In one preferred aspect, the medium comprises and/or is enriched with aprotein. Here, preferably, the protein is either derived from or isderivable from or is in a dairy product, such as milk orcheese—preferably wherein the protein is casing or whey protein—and/orderived from or is derivable from or is in a plant product.

If the beverage is an acidified milk drink, then it is typicallyprepared by acidifying the milk and then adding the pectin at a low pH.

If the beverage is a soya protein drink, then it is typically preparedby solubilising the soya protein at neutral pH. The pectin is added bysolubilization in the soya protein solution at neutral pH. Then, thesolution is acidified by addition e.g. fruit juice.

The use of a block-wise enzymatically de-esterified pectin—which ispreferably prepared by use of recombinant DNA techniques—is of benefitas it allows proteins such as whey and milk proteins (such as casein) tobe stable in acidic solutions. This is of importance for the drinksmarket, such as skimmed milk, fruit juices and whey protein drinks,wherein before it was only possible to retain the flavour of the keyproteins under fairly high acidic conditions—such as pH 4.2—if highamounts of stabiliser were present.

We have now found that for some applications small amounts of thede-esterified pectin prepared by the process of the present inventioncan be employed. At these low levels, the de-esterified pectin accordingto the present invention not only acts as a stabiliser but also it doesnot have an adverse effect on the final product.

If desired, the use of the de-esterified pectin of the present inventionwould enable food manufacturers to increase the pH of foods, such asdrinks. In this regard, in some cases the less acidic nature of thedrinks may make them more palatable for people, especially infants.Thus, in contrast to the prior art processes, it is now possible toretain the flavour of those proteins at pH conditions higher than 4.2,such as up to pH 5.5 (such as pH 5.2) by use of the block-wiseenzymatically de-esterified pectin, particularly the block-wiseenzymatically de-esterified pectin prepared by use of, for example,recombinant DNA techniques.

In addition, it is believed that even under low pH conditions, such aspH 4.2 or less, the block-wise enzymatically de-esterifiedpectin—particularly the block-wise enzymatically de-esterified pectin(preferably prepared by use of recombinant DNA techniques)—stabilisesthe protein(s) more than the prior art stabilisers that are used forthose pH conditions.

A further advantage is that the PME of the present invention is capableof producing a substantially homogeneous block-wise de-esterifiedpectin. By this we mean that substantially all of the pectin chainscomprise at least two adjacent de-esterified carboxyl groups. However,for some applications it may not be necessary to prepare or use such asubstantially homogeneous block-wise de-esterified pectin.

Without wishing to be bound by theory it is believed that the block-wiseenzymatically de-esterified pectin—particularly that prepared by use ofrecombinant DNA techniques—stabilises the protein(s) by surrounding theprotein(s) in a blanket of negative charges, thus forming a stableentity.

The PME enzyme of the present invention is useful for blockwisede-esterifying pectins when the pectins are contacted with the enzyme ina substantially aqueous medium. In some instances, de-esterifyingpectins can increase the calcium ion sensitivity of a pectin—which inturn may be advantageous.

Alternatively, the PME enzyme of the present invention is useful foresterifying pectins when the pectins are contacted with the enzyme in asubstantially non-aqueous medium, such as in the presence of methanol orin the presence of high concentrations of ammonium sulphate. This aspectis advantageous if, for example, it is desirable to reduce the calciumsensitivity of a pectin.

This method of esterifying pectins is advantageous because it obviatesthe need for the high temperature and methanol esterification conditionsassociated with the prior art processes. Thus, the present inventionalso includes the use of that esterified pectin in the preparation of afoodstuff as well as the pectin per se.

In accordance with the present invention, the de-esterified pectin ofthe present invention is advantageous for the preparation of afoodstuff.

Preferably, the foodstuff is food for human and/or animal consumption.Typical preferred foodstuffs include jams, marmalades, jellies, dairyproducts (such as milk or cheese), meat products, poultry products, fishproducts and bakery products. The foodstuff may even be a beverage. Thebeverage can be a drinking yoghurt, a fruit juice or a beveragecomprising whey protein.

In addition to the foodstuff comprising the PME treated pectin, thefoodstuff may comprise more other components, such as one or moresuitable food ingredients. Typical food ingredients include any one ormore of an acid—such as citric acid—or a sugar—such as sucrose, glucoseor invert sugar—or fruit—or other enzymes, preservatives, colourings andother suitable components.

In one preferred embodiment, the foodstuff of the present inventioncomprises fruit. Here, fruit imparts taste, colour and structure to thegel, as well as pectin, acid and a small amount of solids. Depending onthe level of natural flavour and colour in the fruit, fruit dosages arenormally from 25% to 60% of the jam. The solids content of ordinaryfruit is around 10% Brix, but fruit concentrate, which is typically65-70% Brix, can also be used. The pH in fruit varies widely, dependingon the fruit in question, but most fruits have a pH between 3.0 and 3.5.

The pectin content also varies, depending on the fruit in question. Forexample, redcurrants, blackcurrants and oranges have a high pectincontent, and satisfactory gels from these fruits can be obtained byadding only a small amount of extra pectin. The choice of pectin dependson the type of jam in question. For example, GRINDSTED™ Pectin SS 200 isused in jams containing no fruit pieces or jam containing only verysmall fruit pieces. Fruit separation in such jams is not a problem, andconsequently a slow-setting pectin and lower filling temperature can beused.

By way of example, GRINDSTED™ Pectin RS 400 is used in jams containinglarge fruit pieces or whole fruit, for instance cherries orstrawberries. In jams containing whole fruit it may be difficult toavoid fruit separation, and it is therefore necessary to use a rapid-setpectin such as GRINDSTED™ Pectin RS 400.

The choice of pectin type may also depend on the container size inquestion. When standard jars are used, the filling temperature is lesscritical with regard to the stability of pectin, as the jars will cooldown relatively quickly after filling and the pectin sill not degrade.However, if the jam is filled into large containers, eg 500 or 1,000 kg,the cooling time will be very long. In the centre of such a largecontainer the pectin will be especially subject to degradation, and thegel will be weaker at the centre than at the sides. Consequently, a moreslow-setting pectin is generally used for large containers, allowingfilling at lower temperatures and thereby avoiding degradation of thepectin.

Sugar is added to jam for various reasons, such as:

1. To provide soluble solids—HE pectins can require a minimum solublesolids content of 55% before they will gel

2. To provide sweetness

3. To provide increased physical, chemical and microbiological stability

4. To provide an improved mouthfeel

5. To provide improved colour and gloss

Sucrose is the sugar normally used, but other sugars may well be useddepending on the taste, sweetening effect, crystallisation or structurerequired. Price may also influence which type of sugar is used.

Invert sugar has the same sweetening effect as sucrose, whereas glucosesyrup, glucose and sorbitol have a reduced sweetening effect. Highfructose corn syrup and fructose will have a greater sweetening effectthan sucrose.

The structure and strength of the gel as well as the gelling temperaturewill, to some extent, be influenced by changes in sugar composition.

Acid is added for two reasons: 1) partly to reduce the pH level to3.0-3.2 to obtain a satisfactory gel with the pectin, and 2) partly toenhance the flavour of the fruit. The optimum pH for gelation using theHE pectins depends on the type of pectin and solids content in question.

If GRINDSTED™ Pectin SS 200 is used in jam with 65-68% Brix, the optimumpH is 3.0-3.2. If the solids content is higher than this, the optimum pHis 3.1-3.3. Conversely, if the solids content is lower the optimum pH is2.8-3.0. If GRINDSTED™ Pectin RS 400 is used, the optimum pH isapproximately 0.2 units higher than for GRINDSTED™ Pectin SS 200.

The acid most commonly used is citric acid, monohydrate, in a 50% w/vsolution.

Other acids (such as malic acid, tartaric acid or phosphoric acid) maybe used but must always be in solution.

The choice of acid depends on legislation, price, and the tartness ofsweetness required in the finished product.

Citric acid imparts a relatively strong acid taste to the finishedproduct, whereas malic acid results in a softer but longer-lastingtaste.

Tartaric acid may result in a slightly bitter taste, and phosphoric acidresults in a sweeter taste.

Enzymaticallly treated pectin can prevent syneresis which can oftenoccur in the manufacture of marmalades and jams with low soluble solidscontents.

In some instances, the de-esterified pectin of the present invention isalso advantageous for use as a stabiliser and/or viscosity modifier inthe preparation of pharmaceuticals, pharmaceutical appliances, cosmeticsand cosmetic appliances.

Preferably the block-wise enzymatically de-esterified pectin is a highester pectin containing about 80% ester groups or less (i.e. a degree ofesterification (DE) of 80% or less), preferably about 75% ester groupsor less (i.e. a DE of about 75% or less). In this regard, the ratio offree carboxyl groups to esterified carboxyl groups on the pectin is from1:1 to 1:4, preferably from 1:2 to 1:3.

Preferably, the block-wise enzymatically de-esterified pectin containsabout 78% ester groups.

A Protocol for determining the degree of esterification of the PMEsubstrate may be found on page 58 of WO-A-97/03574 (the contents ofwhich are incorporated herein by reference). For ease of reference, thisProtocol is recited after the Examples Section

A Protocol for determining calcium sensitivity may be found on page 57of WO-A-97/03574 (the contents of which are incorporated herein byreference). For ease of reference, this Protocol is also recited afterthe Examples Section.

Preferably the block-wise enzymatically de-esterified pectin has a highmolecular weight. Typically, the molecular weight is between from about50 KD to about 150 KD.

Preferably the block-wise enzymatically de-esterified pectin is preparedby treating a pectin with a PME that de-esterifies two or more adjacentgalacturonic acid residues of the pectin on at least substantially allof the pectin chains.

Preferably the PME is derived from a PME obtainable from amicro-organism, preferably a bacterium.

The term “derived from a PME obtainable from a micro-organism” meansthat the PME has a sequence similar to that of a PME that is obtainablefrom a micro-organism providing the PME can de-esterify pectin in ablock-wise manne

The term “derived from a PME obtainable from a bacterium” means that thePME has a sequence similar to that of a PME that is obtainable from abacterium, providing the PME can de-esterify pectin in a block-wisemanner.

The term “pectin” includes pectin in its normal sense, as well asfractions and derivatives thereof, as well as modified pectins (e.g.chemically modified pectins and enzymatically modified pectins).

By way of example the pectin can be a derivatised pectin, a degraded(such as partially degraded) pectin or a modified pectin. An example ofa modified pectin is pectin that has been prior treated with an enzymesuch as a PME—which may be the same as the PME of the present inventionor a different PME or a combination thereof. An example of a pectinderivative is pectin that has been chemically treated—eg. amidated.

Preferably, the pectin is not a pectin that has been prior treated withthe enzyme polygalacturonase to substantially reduce the length of thepectin backbone.

As indicated, in a preferred aspect, the present invention encompassesvariants, homologues and derivatives of the sequences presented herein.The present invention also encompasses fragments of such sequences.

The terms “variant”, “homologue” or “fragment” in relation to therecombinant enzyme of the present invention include any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acid from or to the sequence providing theresultant amino acid sequence has PME activity, preferably having atleast the same activity of a recombinant enzyme comprising sequenceshown as SEQ I.D. No. 2. In particular, the term “homologue” covershomology with respect to structure and/or function providing theresultant recombinant enzyme has PME activity. With respect to sequencehomology (i.e. similarity), preferably there is at least 75%, morepreferably at least 85%, more preferably at least 90% homology to thesequence shown in the attached sequence listings. More preferably thereis at least 95%, more preferably at least 98%, homology to the sequenceshown in the attached sequence listings.

Thus, enzymes of the present invention may also be modified to containone or more (e.g. at least 2, 3, 5, or 10) substitutions, deletions orinsertions, including conserved substitutions.

By way of example, conservative substitutions may be made, for exampleaccording to the Table below. Amino acids in the same block in thesecond column and preferably in the same line in the third column may besubstituted for each other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

As indicated above, proteins of the invention are typically made byrecombinant means for example as described herein and/or by usingsynthentic means techniques well known to skilled persons such as solidphase synthesis. Variants and derivatives of such sequences includefusion proteins, wherein the fusion proteins comprise at least the aminoacid sequence of the present invention being linked (directly orindirectly) to another amino acid sequence. These other amino acidsequences—which are sometimes referred to as fusion proteinpartners—will typically impart a favourable functionality—such as to aidextraction and purification of the amino acid sequence of the presentinvention. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and β-galactosidase. It may also beconvenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of the present invention so asto allow removal of the latter. Preferably the fusion protein partnerwill not hinder the function of the protein of the present invention.

In one aspect, the variant, homologue, derivative, or fragment of theamino acid sequence according to the present invention may comprise atleast the following domain—which we have presented as Formula (I):

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22  (I)

wherein

A1 is a hydrophobic or polar amino acid or a neutral amino acid

A2 is a hydrophobic amino acid

A3 is a hydrophobic amino acid

A4 is a polar amino acid

A5 is a polar or charged amino acid or neutral amino acid

A6 is a polar amino acid

A7 is a polar or charged or hydrophobic amino acid

A8 is a hydrophobic amino acid

A9 is a hydrophobic or polar amino acid

A10 is a hydrophobic or polar amino acid

A11 is a charged amino acid

A12 is a charged or polar or hydrophobic amino acid

A13 is a hydrophobic or charged amino acid or neutral amino acid

A14 is a hydrophobic or polar amino acid or charged or neutral aminoacid

A15 is a charged or polar or hydrophobic amino acid

A16 is a polar or hydrophobic or charged amino acid or neutral aminoacid

A17 is a polar or charged amino acid or neutral amino acid

A18 is a polar or charged or hydrophobic amino acid

A19 is a polar amino acid or a neutral amino acid

A20 is a hydrophobic or polar amino acid

A21 is a hydrophobic amino acid

A22 is a polar or hydrophobic amino acid.

This domain is described in our earlier UK patent application No.9910935.7 filed May 11, 1999.

In this aspect of the present invention, preferably, A1 is a hydrophobicamino acid.

Preferably A5 is a polar amino acid.

Preferably A7 is a polar amino acid.

Preferably A9 is a hydrophobic amino acid.

Preferably A10 is a hydrophobic amino acid.

Preferably A12 is a charged amino acid.

Preferably A13 is a hydrophobic amino acid.

Preferably A14 is a hydrophobic amino acid.

Preferably A15 is a charged amino acid.

Preferably A16 is a polar amino acid.

Preferably A17 is a polar amino acid.

Preferably A18 is a polar amino acid.

Preferably A20 is a hydrophobic amino acid.

Preferably A22 is a polar amino acid.

For the amino acid sequence of formula (I), preferable examples ofhydrophobic amino acids may include: Ala (A), Val (V), Phe (F), Pro (P),Met (M), Ile (I), Leu (L).

For the amino acid sequence of formula (I), preferable examples ofcharged amino acids may include Asp (D), Glu (E), Lys (K), Arg (R).

For the amino acid sequence of formula (I), preferable examples of polaramino acids may include: Ser (S), Thr (T), Tyr (Y), His (H), Cys (C),Asn (N), Gln (Q), Trp (W).

For the amino acid sequence of formula (I), a preferable example of aneutral amino acid is glycine (G).

Preferably A1 is A, V, G or T.

Preferably A2 is V or L.

Preferably A3 is L, F or I.

Preferably A4 is Q.

Preferably A5 is N, D, K, G or S.

Preferably A6 is C or S.

Preferably A7 is D, Q, K, E, Y or L.

Preferably A8 is I, L or F.

Preferably A9 is H, N, V, M or L.

Preferably A10 is A, C, I, P, L, C or S.

Preferably A11 is R.

Preferably A12 is K, R, L, Q or Y.

Preferably A13 is P, G or R.

Preferably A14 is N, G, M, A, L, R or S.

Preferably Al5 is S, K, E, P or D.

Preferably A16 G, Y, H, N, K or V.

Preferably A17 is Q, G or K.

Preferably A18 is K, Q, F, Y, T or S.

Preferably A19 is N, C or G.

Preferably A20 is M, L, I, T, V, H or N.

Preferably A21 is V or I.

Preferably A22 is T, L or S.

We also believe that the amino acid sequence should retain the aminoacid of sequence presented as formula (II):

N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-H-H-N-H-N-N-N-N-N-N-N-N-N-N-N-N-N-H-N-N-N-P-C-P-H-N-H-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-H-N-G-N-N-C-N-H-H-G-N-N-N  (II)

wherein

H independently represents a hydrophobic amino acid

C independently represents a charged amino acid

P independently represents a polar amino acid

G represents glycine

N independently represents glycine or a hydrophobic or charged or polaramino acid.

For the amino acid sequence of formula (II): examples of hydrophobicamino acids include: Ala (A), Val (V), Phe (F), Pro (P), Met (M), lie(I), Leu (L); examples of charged amino acids include Asp (D), Glu (E),Lys (K), Arg (R); and examples of polar amino acids include: Ser (S),Thr (T), Tyr (Y), His (H), Cys (C), Asn (N), Gln (Q), Trp (W).

The terms “variant”, “homologue” or “fragment” in relation to thenucleotide sequence coding for the recombinant enzyme of the presentinvention include any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) nucleic acidfrom or to the sequence providing the resultant nucleotide sequencecodes for a recombinant enzyme having PME activity, preferably having atleast the same activity of a recombinant enzyme comprising the sequenceshown as SEQ I.D. No. 2. In particular, the term “homologue” covershomology with respect to structure and/or function providing theresultant nucleotide sequence codes for a recombinant enzyme having PMEactivity. With respect to sequence homology (i.e. similarity),preferably there is at least 75%, more preferably at least 85%, morepreferably at least 90% homology. More preferably there is at least 95%,more preferably at least 98%, homology.

In a preferred aspect the terms “variant”, “homologue” or “fragment” inrelation to the nucleotide sequence coding for the recombinant enzyme ofthe present invention include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence sequence shown as SEQ I.D.No. 1 providing the resultant nucleotide sequence codes for arecombinant enzyme having PME activity, preferably having at least thesame activity of a recombinant enzyme comprising the sequence shown asSEQ I.D. No. 2. In particular, the term “homologue” covers homology withrespect to structure and/or function providing the resultant nucleotidesequence codes for a recombinant enzyme having PME activity. Withrespect to sequence homology (i.e. similarity), preferably there is atleast 75%, more preferably at least 85%, more preferably at least 90%homology. More preferably there is at least 95%, more preferably atleast 98%, homology.

The above terms are synonymous with allergic variations of thesequences.

As indicated above, the present invention concerns the sequencepresented in the attached sequence listings, or a variant, derivative orhomologue thereof.

Preferably, the variant, derivative or homologue can have at least 75%sequence homology (i.e. identity) with any one or more of the sequencespresented.

In particular, the term “homology” as used herein may be equated withthe term “identity”.

Here, sequence homology can be determined by a simple “eyeball”comparison of any one or more of the sequences with another sequence tosee if that other sequence has at least 75% identity to the sequence(s).

Relative sequence homology (i.e. sequence identity) can also bedetermined by commercially available computer programs that cancalculate % homology between two or more sequences. A typical example ofsuch a computer program is CLUSTAL.

Sequence homology (or identity) may moreover be determined using anysuitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andare advantageously set to the defined default parameters.

Advantageously, “substantial homology” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (seehttp://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.The BLAST programs were tailored for sequence similarity searching, forexample to identify homologues to a query sequence. The programs are notgenerally useful for motif-style searching. For a discussion of basicissues in similarity searching of sequence databases, see Altschul et al(1994) Nature Genetics 6:119-129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov performthe following tasks:

blastp compares an amino acid query sequence against a protein sequencedatabase;

blastn compares a nucleotide query sequence against a nucleotidesequence database;

blastx compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase;

tblastn compares a protein query sequence against a nucleotide sequencedatabase dynamically translated in all six reading frames (bothstrands).

tblastx compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page). See also EXPECT andCUTOFF.

ALIGNMENTS Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).The valid alternative choices include: PAM40, PAM120, PAM250 andIDENTITY. No alternate scoring matrices are available for BLASTN;specifying the MATRIX directive in BLASTN requests returns an errorresponse.

STRAND Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Claverie & States (1993) Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g., hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g. “XXXXXXXXX”).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect.

Furthermore, in some cases, sequences are masked in their entirety,indicating that the statistical significance of any matches reportedagainst the unfiltered query sequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

For some applications, preferably sequence comparisons are conductedusing the simple BLAST search algorithm provided athttp://www.ncbi.nlm.nih.gov/BLAST.

Should Gap Penalties be used when determining sequence identity, thenpreferably the following parameters are used:

FOR BLAST GAP OPEN 0 GAP EXTENSION 0 FOR CLUSTAL DNA PROTEIN WORD SIZE 21 K triple GAP PENALTY 10 10 GAP EXTENSION 0.1 0.1

Other computer program methods to determine identify and similaritybetween the two sequences include but are not limited to the GCG programpackage (Devereux et al 1984 Nucleic Acids Research 12: 387 and FASTA(Atschul et al 1990 J Molec Biol 403-410).

The present invention also encompasses nucleotide sequences that arecomplementary to the sequences presented herein, or any derivative,fragment or derivative thereof. If the sequence is complementary to afragment thereof then that sequence can be used a probe to identifysimilar coding sequences in other organisms etc.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences presented herein, or anyderivative, fragment or derivative thereof.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences that are complementary to thesequences presented herein, or any derivative, fragment or derivativethereof.

The term “complementary” also covers nucleotide sequences that canhybridise to the nucleotide sequences of the coding sequence.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hydridising understringent conditions (eg. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.) as well as the process of amplification as carriedout in polymerase chain reaction technologies as described inDieffenbach C W and G S Dveksler (1995, PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview N.Y.).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex, as taught inBerger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methodsin Enzymology, Vol 152, Academic Press, San Diego Calif.), and confer adefined “stringency” as explained below.

Maximum stringency typically occurs at about Tm−5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC).

The term “nucleotide” in relation to the present invention includesgenomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, morepreferably cDNA for the coding sequence of the present invention.

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes the nucleotide sequence according tothe present invention or, the case of the combination of constructs, theGOI directly or indirectly attached to a promoter. An example of anindirect attachment is the provision of a suitable spacer group such asan intron sequence such as the Sh1-intron or the ADH intron,intermediate the promoter and the nucleotide sequence of the presentinvention or the GOI. The same is true for the term “fused” in relationto the present invention which includes direct or indirect attachment.In each case, the terms do not cover the natural combination of the genecoding for the enzyme ordinarily associated with the wild type genepromoter and when they are both in their natural environment.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a filamentousfungus, preferably of the genus Aspergillus, such as Aspergillus niger,or plants, such as potatoes, sugar beet etc., into which it has beentransferred. Various markers exist which may be used, such as forexample those encoding mannose-6-phosphate isomerase (especially forplants) or those markers that provide for antibiotic resistance—e.g.resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.

The term “vector” includes expression vectors and transformationvectors.

The term “expression vector” means a construct capable of in vivo or invitro expression.

The term “transformation vector” means a construct capable of beingtransferred from one species to another—such as from an E.coli plasmidto a filamentous fungus, preferably of the genus Aspergillus. It mayeven be a construct capable of being transferred from an E. coli plasmidto an Agrobacterium to a plant.

The term “tissue” includes tissue per se and organ.

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence coding for therecombinant enzyme according to the present invention and/or productsobtained therefrom, wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe organism.

Preferably the organism is a filamentous fungus, preferably of the genusAspergillus, more preferably Aspergillus niger.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence coding forthe recombinant enzyme according to the present invention and/orproducts obtained therefrom, wherein the promoter can allow expressionof the nucleotide sequence according to the present invention within theorganism. Preferably the nucleotide sequence is incorporated in thegenome of the organism.

Preferably the transgenic organism is a filamentous fungus, preferablyof the genus Aspergillus, more preferably Aspergillus niger.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, a promoter, thenucleotide sequence coding for the recombinant enzyme according to thepresent invention, constructs according to the present invention(including combinations thereof), vectors according to the presentinvention, plasmids according to the present invention, cells accordingto the present invention, tissues according to the present invention orthe products thereof.

The term “transgenic organism” does not cover the native nucleotidecoding sequence according to the present invention in its naturalenvironment when it is under the control of its native promoter which isalso in its natural environment. In addition, the present invention doesnot cover the native enzyme according to the present invention when itis in its natural environment and when it has been expressed by itsnative nucleotide coding sequence which is also in its naturalenvironment and when that nucleotide sequence is under the control ofits native promoter which is also in its natural environment.

The transformed cell or organism could prepare acceptable quantities ofthe desired compound which would be easily retrievable from, the cell ororganism.

Preferably the construct of the present invention comprises thenucleotide sequence of the present invention and a promoter.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

In one aspect, the nucleotide sequence according to the presentinvention is under the control of a promoter that may be a cell ortissue specific promoter. If, for example, the organism is a plant thenthe promoter can be one that affects expression of the nucleotidesequence in any one or more of tuber, stem, sprout, root and leaftissues.

By way of example, the promoter for the nucleotide sequence of thepresent invention can be the α-Amy 1 promoter (otherwise known as theAmy 1 promoter, the Amy 637 promoter or the α-Amy 637 promoter) asdescribed in our co-pending UK; patent application No. 9421292.5 filedOct. 21, 1994. Alternatively, the promoter for the nucleotide sequenceof the present invention can be the α-Amy 3 promoter (otherwise known asthe Amy 3 promoter, the Amy 351 promoter or the α-Amy 351 promoter) asdescribed in our co-pending UK patent application No. 9421286.7 filedOct. 21, 1994.

The promoter could additionally include features to ensure or toincrease expression in a suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box. The promoter mayeven contain other sequences to affect (such as to maintain, enhance,decrease) the levels of expression of the nucleotide sequence of thepresent invention or, in the case of the combination of constructs, theGOI. For example, suitable other sequences include the Sh1-intron or anADH intron. Other sequences include inducible elements—such astemperature, chemical, light or stress inducible elements. Also,suitable elements to enhance transcription or translation may bepresent. An example of the latter element is the TMV 5′ signal sequence(see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23[1993] 97).

In addition the present invention also encompasses combinations ofpromoters and/or nucleotide sequences coding for proteins or recombinantenzymes and/or elements.

The present invention also encompasses the use of promoters to express anucleotide sequence coding for the recombinant enzyme according to thepresent invention or the GOI, wherein a part of the promoter isinactivated but wherein the promoter can still function as a promoter.Partial inactivation of a promoter in some instances is advantageous. Inparticular, with the Amy 351 promoter mentioned earlier it is possibleto inactivate a part of it so that the partially inactivated promoterexpresses the nucleotide of the present invention or a GOI in a morespecific manner such as in just one specific tissue type or organ.

The term “inactivated” means partial inactivation in the sense that theexpression pattern of the promoter is modified but wherein the partiallyinactivated promoter still functions as a promoter. However, asmentioned above, the modified promoter is capable of expressing thenucleotide of the present invention or a GOI in at least one (but notall) specific tissue of the original promoter. One such promoter is theAmy 351 promoter described above. Examples of partial inactivationinclude altering the folding pattern of the promoter sequence, orbinding species to parts of the nucleotide sequence, so that a part ofthe nucleotide sequence is not recognised by, for example, RNApolyrnerase. Another, and preferable, way of partially inactivating thepromoter is to truncate it to form fragments thereof. Another way wouldbe to mutate at least a part of the sequence so that the RNA polymerasecan not bind to that part or another part. Another modification is tomutate the binding sites for regulatory proteins for example the CreAprotein known from filamentous fungi to exert carbon cataboliterepression, and thus abolish the catabolite repression of the nativepromoter.

The term “GOI” with reference to the combination of constructs accordingto the present invention means any gene of interest. A GOI can be anynucleotide that is either foreign or natural to the organisin (e.g.filarnentous fungus preferably of the genus Aspergillus, or a plant) inquestion. Typical examples of a GOI include genes encoding for proteinsand enzymes that modify metabolic and catabolic processes. The GOI maycode for an agent for introducing or increasing pathogen resistance. TheGOI may even be an antisense construct for modifying the expression ofnatural transcripts present in the relevant tissues. The GOI may evencode for a non-native protein of a filamentous fungus, preferably of thegenus Aspergillus, or a compound that is of benefit to animals orhumans.

Examples of GOIs include other pectinases, galactonases, pectindepolymerases, polygalacturonases, pectate lyases, pectin lyases,rhamno-galacturonases, hemicellulases, endo-β-glucanases, arabinases, oracetyl esterases, or combinations thereof, as well as antisensesequences thereof.

These other types of enzymes can be added at the same time as the PMEor, alternatively, prior to or after the addition of the PME.

By way of example, the GOI can be a PME as disclosed in WO-A-97/03574 orthe PME disclosed in either WO-A-94/25575 or WO-A-97/31102 as well asvariants, derivatives or homologues of the sequences disclosed in thosepatent applications.

The GOI may be a protein giving nutritional value to a food or crop.Typical examples include plant proteins that can inhibit the formationof anti-nutritive factors and plant proteins that have a more desirableamino acid composition (e.g. a higher lysine content than anon-transsenic plant). The GOI may even code for an enzyme that can beused in food processing such as chymosin, thaumatin and α-galactosidase.The GOI can be a gene encoding for any one of a pest toxin, an antisensetranscript such as that for patatin or α-amylase, ADP-glucosepyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, aglucanase or genomic PME.

The GOI may even code for an intron of a particular enzyme but whereinthe intron can be in sense or antisense orientation. In the latterinstance, the particular enzyme could be genomic PME. Antisenseexpression of enomic exon or intron sequences as the GOI would mean thatthe natural PME expression would be reduced or eliminated but whereinthe recombinant PME expression would not be affected. This isparticularly true for antisense intron or sense intron expression.

The GOI can be the nucleotide sequence coding for the α-amylase enzymewhich is the subject of our co-pending UK patent application 9413439.2filed on Jul. 4, 1994. The GOI can be the nucleotide sequence coding forthe α-amylase enzyme which is the subject of our UK patent application9421290.9 filed on Oct. 21, 1994. The GOI can be any of the nucleotidesequences coding for the ADP-glucose pyrophosphorylase enzymes which arethe subject of our PCT patent application PCT/EP94/01082 filed Apr. 7,1994. The GOI can be any of the nucleotide sequences coding for theα-glucan lyase enzyme which are described in our PCT patent applicationPCT/EP94/03397 filed Oct. 15, 1994.

The host organism can be a prokaryotic or a eukaryotic organism.Examples of suitable prokaryotic hosts include E. coli and Bacillussubtilis. Teachings on the transformation of prokaryotic hosts is welldocumented in the art, for example see Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring HarborLaboratory Press). If a prokaryotic host is used then the gene may needto be suitably modified before transformation—such as by removal ofintrons.

As mentioned above, a preferred host organism is of the genusAspergilus, such as Aspergillus niger.

A transsgenic Aspergillus according to the present invention can beprepared by following the teachings of Rambosek, J. and Leach, J. 1987(Recombinant DNA in filamentous fungi: Progress and Prospects, CRC Crit.Rev. Biotechnol. 6:357-393), Davis R. W. 1994 (Heterologous geneexpression and protein secretion in Aspergillus. In: Martinelli S. D.,Kinghorn J. R.(Editors) Aspergilius: 50 years on. Progress in industrialmicrobiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview ofFungal Gene structure. In: Leong, S. A., Berka R. M. (Editors) MolecularIndustrial Mycology. Systems and Applications for Filamentous Fungi.Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectorsfor genetic manipulation. In: Martinelli S. D. Kinghorn J. R.(Editors)Aspergillus: 50 years on. Progress in industrial microbiology vol 29.Elsevier Amsterdam 1994. pp. 641-666). However, the following commentaryprovides a summary of those teachings for producing transgenicAspergillus according to the present invention.

For almost a century, filamentous fungi have been widely used in manytypes of industry for the production of organic compounds and enzymes.For example, traditional japanese koji and soy fermentations have usedAspergillus sp. Also, in this century Aspergillus niger has been usedfor production of organic acids particular citric acid and forproduction of various enzymes for use in industry.

There are two major reasons why filamentous fungi have been sowidelyused in industry. First filamentous fungi can produce high amountsof extracelluar products, for example enzymes and organic compounds suchas antibiotics or organic acids. Second filamentous fungi can grow onlow cost substrates such as grains, bran, beet pulp etc. The samereasons have made filamentous fungi attractive organisms as hosts forheterologous expression according to the present invention.

In order to prepare the transgenic Aspergillus, expression constructsare prepared by inserting the nucleotide sequence according to thepresent invention (or even the GOI) into a construct designed forexpression in filamentous fungi.

Several types of constructs used for heterologous expression have beendeveloped. These constructs preferably contain a promoter which isactive in fungi. Examples of promoters include a fungal promoter for ahighly expressed extracelluar enzyme, such as the glucoamylase promoteror the α-amylase promoter. The nucleotide sequence according to thepresent invention (or even the GOI) can be fused to a signal sequencewhich directs the protein encoded by the nucleotide sequence accordingto the present invention (or even the GOI) to be secreted. Usually asignal sequence of fungal origin is used. A terminator active in fungiends the expression system.

Another type of expression system has been developed in fungi where thenucleotide sequence according to the present invention (or even the GOI)can be fused to a smaller or a larger part of a fungal gene encoding astable protein. This can stabilize the protein encoded by the nucleotidesequence according to the present invention (or even the GOI). In such asystem a cleavage site, recognized by a specific protease, can beintroduced between the fungal protein and the protein encoded by thenucleotide sequence according to the present invention (or even theGOI), so the produced fusion protein can be cleaved at this position bythe specific protease thus liberating the protein encoded by thenucleotide sequence according to the present invention (or even theGOI). By way of example, one can introduce a site which is recognized bya KEX-2 like peptidase found in at least some Aspergilli. Such a fusionleads to cleavage in vivo resulting in protection of the expressedproduct and not a larger fusion protein.

Heterologous expression in Aspergillus has been reported for severalgenes coding for bacterial, fungal, vertebrate and plant proteins. Theproteins can be deposited intracellularly if the nucleotide sequenceaccording to the present invention (or even the GOI) is not fused to asignal sequence. Such proteins will accumulate in the cytoplasm and willusually not be glycosylated which can be an advantage for some bacterialproteins. If the nucleotide sequence according to the present invention(or even the GOI) is equipped with a signal sequence the protein willaccumulate extracelluarly.

With regard to product stability and host strain modifications someheterologous proteins are not very stable when they are secreted intothe culture fluid of fungi. Most fungi produce several extracelluarproteases which degrade heterologous proteins. To avoid this problemspecial fungal strains with reduced protease production have been usedas host for heterologous production.

For the transformation of filamentous fungi, several transformationprotocols have been developed for many filamentous fungi (Ballance 1991,ibid). Many of them are based on preparation of protoplasts andintroduction of DNA into the protoplasts using PEG and Ca²⁺ ions. Thetransformed protoplasts then regenerate and the transformed fungi areselected using various selective markers. Among the markers used fortransformation are a number of auxotrophic markers such as argB, trpC,niaD and pyrG, antibiotic resistance markers such as benomyl resistance,hygromycin resistance and phleomycin resistance. A commonly usedtransformation marker is the amds gene of A. nidulans which in high copynumber allows the fungus to grow with acrylamide as the sole nitrogensource.

In another embodiment the transgenic organism can be a yeast. In thisregard, yeast have also been widely used as a vehicle for heterologousgene expression. The species Saccharomyces cerevisiae has a long historyof industrial use, including its use for heterologous gene expression.Expression of heterologous genes in Saccharomyces cerevisiae has beenreviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al,eds, pp 401-429, Allen and Unwin, London) and by King et al (1989,Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds,pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrativevectors, which require recombination with the host genome for theirmaintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructsare prepared by inserting the nucleotide sequence of the presentinvention into a construct designed for expression in yeast. Severaltypes of constructs used for heterologous expression have beendeveloped. The constructs contain a promoter active in yeast fused tothe nucleotide sequence of the present invention, usually a promoter ofyeast origin, such as the GAL1 promoter, is used. Usually a signalsequence of yeast origin, such as the sequence encoding the SUC2 signalpeptide, is used. A terminator active in yeast ends the expressionsystem.

For the transformation of yeast several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al (1978, Proceedings of the National Academy of Sciences of the USA75, 1929); Beggs, J D (1978, Nature, London, 275. 104): and Ito. H et al(1983, J Bacteriology 153, 163-168).

The transformed yeast cells are selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, eg G418.

Another host organism is a plant.

Even though the enzyme and the nucleotide sequence coding therefor arenot disclosed in EP-B-0470145 and CA-A-2006454, those two documents doprovide some useful background commentary on the types of techniquesthat may be employed to prepare transgenic plants according to thepresent invention. Some of these background teachings are now includedin the following commentary.

The basic principle in the construction of genetically modified plantsis to insert genetic information in the plant genome so as to obtain astable maintenance of the inserted genetic material.

Several techniques exist for inserting the genetic information, the twomain principles being direct introduction of the genetic information andintroduction of the genetic information by use of a vector system. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27).

Thus, in one aspect, the present invention relates to a vector systemwhich carries a nucleotide sequence or construct according to thepresent invention and which is capable of introducing the nucleotidesequence or construct into the genome of an organism, such as a plant.

The vector system may comprise one vector, but it can comprise twovectors. In the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells with agiven promoter or nucleotide sequence or construct is based on the useof a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid fromAgrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305and Butcher D. N. et al. (1980), Tissue Culture Methods for PlantPathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208.

Several different Ti and Ri plasmids have been constructed which aresuitable for the construction of the plant or plant cell constructsdescribed above. A non-limiting example of such a Ti plasmid is pGV3850.

The nucleotide sequence or construct of the present invention shouldpreferably be inserted into the Ti-plasmid between the terminalsequences of the T-DNA or adjacent a T-DNA sequence so as to avoiddisruption of the sequences immediately surrounding the T-DNA borders,as at least one of these regions appear to be essential for insertion ofmodified T-DNA into the plant genome.

As will be understood from the above explanation, if the organism is aplant, then the vector system of the present invention is preferably onewhich contains the sequences necessary to infect the plant (e.g. the virregion) and at least one border part of a T-DNA sequence, the borderpart being located on the same vector as the genetic construct.Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmidor an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, asthese plasmids are well-known and widely employed in the construction oftransgenic plants, many vector systems exist which are based on theseplasmids or derivatives thereof.

In the construction of a transgenic plant the nucleotide sequence orconstruct of the present invention may be first constructed in amicroorganism in which the vector can replicate and which is easy tomanipulate before insertion into the plant. An example of a usefulmicroorganism is E. coli., but other microorganisrns having the aboveproperties may be used. When a vector of a vector system as definedabove has been constructed in E. coli. it is transferred, if necessary,into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.The Ti-plasmid harbouring the nucleotide sequence or construct of theinvention is thus preferably transferred into a suitable Agrobacteriumstrain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cellharbouring the nucleotide sequence or construct of the invention, whichDNA is subsequently transferred into the plant cell to be modified.

As reported in CA-A-2006454, a large amount of cloning vectors areavailable which contain a replication system in E. coli and a markerwhich allows a selection bf the transformed cells. The vectors containfor example pBR 322, the pUC series, the M13 mp series, pACYC 184 etc.

In this way, the nucleotide or construct of the present invention can beintroduced into a suitable restriction position in the vector. Thecontained plasmid is used for the transformation in E.coli. The E.colicells are cultivated in a suitable nutrient medium and then harvestedand lysed. The plasmid is then recovered. As a method of analysis thereis generally used sequence analysis, restriction analysis,electrophoresis and further biochemical-molecular biological methods.After each manipulation, the used DNA sequence can be restricted andconnected with the next DNA sequence. Each sequence can be cloned in thesame or different plasmid.

After each introduction method of the desired promoter or construct ornucleotide sequence according to the present invention in the plants thepresence and/or insertion of further DNA sequences may be necessary. If,for example for the transformation the Ti- or Ri-plasmid of the plantcells is used, at least the right boundary and often however the rightand the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areasof the introduced genes, can be connected. The use of T-DNA for thetransformation of plant cells has been intensively studied and isdescribed in EP-A-120516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985. Chapter V; Fraley.et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985)4:277-284.

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al. (1980). Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson. 203-208. For further teachings on thistopic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 199417-27). With this technique, infection of a plant may be done on acertain part or tissue of the plant, i.e. on a part of a leaf, a root, astem or another part of the plant.

Typically, with direct infection of plant tissues by Agrobacteriumcarrying the promoter and/or the GOI, a plant to be infected is wounded,e.g. by cutting the plant with a razor or puncturing the plant with aneedle or rubbing the plant with an abrasive. The wound is theninoculated with the Agrobacterium. The inoculated plant or plant part isthen grown on a suitable culture medium and allowed to develop intomature plants.

When plant cells are constructed, these cells may be grown andmaintained in accordance with well-known tissue culturing methods suchas by culturing the cells in a suitable culture medium supplied with thenecessary growth factors such as amino acids, plant hormones, vitamins,etc. Regeneration of the transformed cells into genetically modifiedplants may be accomplished using known methods for the regeneration ofplants from cell or tissue cultures, for example by selectingtransformed shoots using an antibiotic and by subculturing the shoots ona medium containing the appropriate nutrients, plant hormones, etc.

Further teachings on plant transformation may be found in EP-A-0449375.

The process of the present invention can occur ex vivo or even invivo—such as in planta. In the latter respect, the plant may be atransgenic plant, such as a plant that has been genetically engineeredto produce different levels and/or types of pectin. The plant may alsobe plant material, rather than a whole plant. Here, the plant materialmay be obtained from a transgenic plant, such as a plant that has beengenetically engineered to produce different levels and/or types ofpectin. The plant or plant material may be or may be derived from avegetable, a fruit or other type of pectin containing or producingplant. Here, the vegetable material and/or the fruit material can be amash.

In summation, the present invention provides a process for treating apectin with a pectin methyl esterase (PME); wherein the PME is not aplant PME; but wherein the PME is capable of exhibiting at least oneplant PME property; and wherein the at least one plant PME propertycomprises at least block-wise de-esterification of the pectin.

PME activity itself can be determined quite readily. A protocol fordetermining PME activity is presented after the Examples Section.

The purity of the PME fraction can be investigated by SDS-PAGE usingPharmacia PhastSystem™ with 10-15% SDS-gradient gels. Electrophoresisand silver staining of the proteins can be done as described by themanuals from Pharmacia. For determination of pI IEF 3-9 PhastSystem™gels can be used.

Immuno gel electrophoresis can be used for characterisation ofantibodies (see later section)—such as polyclonal antibodies—raisedagainst PME. The enzyme fractions are then separated on SDS-PAGE andtransferred to NC-paper by semi-dry blotting technique on a Semidrytransfer unit of the PhastSystem™. The NC-paper is incubated with theprimer antibody diluted 1:50 and stained with the second antibodycoupled to alkaline phosphatase (Dako A/S Glsotrup, Denmark) used in adilution of 1:1000.

Further studies that can be performed on the PME include peptidemapping. In this respect, PME can be digested with either trypsin orendo-proteinase Lys-C from Lysobacter enzymogenes (both enzymepreparations should be are sequencing grade)—which can be purchased fromBoerhinger Mannheim, Germany.

Typically, 100 mg purified PME is carboxy methylated with iodoacetamideto protect the reduced SH-groups. Then the protein is cleaved withtrypsin (4 mg/20-100 ml). The hydrolytic cleavage is performed at 40° C.for 2×3 hrs. The reaction is stopped with addition of 20 ml TFA. Aftercentrifugation at 15,000 rpm for 5 min the peptides are purified on areverse-phase HPLC column (Vydac 10 C18 column). 2×500 ml samples areapplied. The peptides are eluted and separated with an increasingacetonitrile gradient from 0.05-0.35% in 60 min in 0.1% TFA. Thepeptides are collected manually in Eppendorf tubes.

For digestion with endo-proteinase Lys-C, freeze dried PME (0.1 mg) isdissolved in 50 ml of 8 M urea, 0.4 M NH₄HCO₃, pH 8.4. After overlaywith N₂ and addition of 5 ml of 45 mM DTT, the protein is denatured andreduced for 15 min at 50° C. under N₂. After cooling to roomtemperature, 5 ml of 100 mM iodoacetamide is added for the cysteines tobe derivatised for 15 min at room temperature in the dark under N₂.Subsequently, 90 ml of water and 5 mg of endo-proteinase Lys-C in 50 ml50 mM tricine and 10 mM EDTA, pH 8.0, are added and the digestion wascarried out for 24 hrs at 37° C. under N₂.

The resulting peptides are then separated as described for trypsindigested peptides.

Selected peptides can be further purified on a Devosil 3 C₁₈ RP-HPLCcolumn 0.46×10 cm (Novo Nordisk, Denmark). The purified peptides arethen applied on an amino acid sequencer, Applied Biosystems 476A, usingpulsed-liquid fast cycles.

Antibodies can be raised against the enzyme of the present invention byinjecting rabbits with the purified enzyme and isolating theimmunoglobulins from antiserum according to procedures describedaccording to N Harboe and A Ingild (“Immunization, Isolation ofImmunoglobulins, Estimation of Antibody Titre” In A Manual ofQuantitative Immunoelectrophoresis, Methods and Applications, N HAxelsen, et al (eds.), Universitetsforlaget. Oslo, 1973) and by T GCooper (“The Tools of Biochemistry”, John Wiley & Sons, New York, 1977).

The present invention will now be described only by way of example, inwhich reference is made to the following attached Figures:

FIG. 1—which is a representation of a plasmid,

FIG. 2—which is a representation of a plasmid,

FIG. 3—which is a representation of a plasmid,

FIG. 4—which is a representation of a plasmid, and

FIG. 5—which is a representation of a plasmid.

EXPERIMENTAL SECTION Cloning of the Erwinia chrysanthemi PME Gene

Materials

Erwinia chrysanthemi (PD97) (“E.chr.”) was purchased from CultureCollection of Plant Protection Service (PD) Wageningen, the Netherlands.

Growth of the Erwinia strain.

The strain was grown in LB-media at 30° C.

DNA

Genomic DNA was isolated from the strain using Qiagen RNA/DNA kit(Qiagen).

PCR

Genomic DNA was used as template.

Primers to clone the PME gene from Erwinia chrysanthemi was designedfrom the published nucleotide sequence of the Erwinia chrysanthemi B374PME gene, accessed from the EMBL/GenBank Data Libraries (accessionnumber Y00549).

Erwinia chrysanthemi PCR primers

PME 5′ end primer: 5′-AGTCGACGTGTATGTTAAAAACGATCTCTGG-3′ (SEQ ID NO:4)

PME 3′ end primer: 5′-AGCGGCCGCAATTCGTCAGGGTAATGTCGG-3 (SEQ ID NO:5)

The 5′ end primers contain a SalI enzyme restriction site, which isWritten in italic and the 3′ end primers contain a Notl enzymerestriction site, which is underlined, in order to facilitate cloning ofthe amplified gene into expression vectors.

PCR was performed with the Expand High Fidelity PCR system (BoehringerMannheim) according to the manufactures instructions with the followingtemperature cycling:

95° C. 3 min in 1 cycle 94° C. 15 sec 50° C. 30 sec 68° C. 3 min in 10cycles 94° C. 15 sec 57° C. 30 sec 68° C. 3 min and additional 20 secpr. cycle in 20 cycles

Cloning of PCR Fragments

PCR fragments were cloned into the PCR 2.1-TOPO cloning vector(Invitrogern), as described by the manufacturer.

DNA Sequencing.

Double stranded DNA was sequenced essentially according to the dideoxymethod of Sanger et al (1979) using the Thermo Sequenase fluorescentlabelled primer cycle sequencing kit with 7-deaza-dGTP (AmnershamPharmacia Biotech), 5′Cy5-labelled primers and the Pharmacia LKB A.L.F.DNA sequencer (Ref: Sanger, F., Nicklen. S., and Coulson A. R. (1979)DNA sequencing with chain-determination inhibitors. Proc. Nat. Acad.Sci. USA 74: 5463-5467). The primers used for sequencing are listedbelow, presented 5′ to 3′:

UNI (M13-20 primer)—17 mer

Cy5-GTAAACGACGGCCAGT (SEQ ID NO:8)

REV—19 mer

Cy5-GGAAACAGCTATGACCATG (SEQ ID NO:7)

E.chr PME—01 17 mer

Cy5-GATTATCCATGCTGGTG (SEQ ID NO:8)

Erwinia chrysanthemi PME—02 18 mer

Cy5-CGGCGTCTATAATGAACG (SEQ ID NO:9)

Erwinia chrysanthemi PME—03 16 mer (SEQ ID NO:10)

Cy5-GCGACAGCGACAGCAG

Erwinia chrysanthemi PME—04 19 mer

Cy5-CCGTGGCAGCCGCAATGAC (SEQ ID NO:11)

The sequenced nucleotide sequence of the PME gene is shown in theattached sequence listings.

EXPRESSION OF THE Erwinia chrysanthemi PME GENE IN E.coli

Generation of the expression vector pATP1:

pATP1 was generated by modifying the pQE60 expression vector from Qiagenin order to use the cloned genes own translation start site and to avoidthe histidine tag. The pQE60 expression vector is shown in FIG. 1.

The 64 bp EcoRI-HindIII fragment was excised from the pQE60 expressionvector and replaced with the 50bp EcoRI-BamHI fragment from the pSPORT1vector (Gibco/BRL), to introduce more enzyme restriction sites.

50 bp pSPORT1 EcoRI-HindIII fragment:

5′ AAGCTTGGATCCTCTAGAGCGGCCGCCGACTAGTGAGCTCGTCGACCCGGAATTC 3′ (SEQ IDNO:12)

The EcoRI enzyme restriction site is underlined and the HindIII enzymerestriction site is written in bold and italic letters.

The 10 bp EcoRI-SalI fragment within the 50 bp fragment obtained fromthe pSPORT1 vector, was replaced with a 28 bp EcoRI-SalI fragmentcontaining a Ribosome Binding Site (RBS) and thereby creating expressionvector pATP1, see FIG. 2.

Generation of the 28 bp EcoRI-SalI fragment by annealing of two oligonucleotides:

RBS primer 1

5′ CACACAGAATTCATTAAAGAGGAGAAATTAACCCGTCGACCCGGGAG 3′ (SEQ ID NO:13)

RBS primer 2:

5′ CTCCCGGGTCGACGGGTTAATTTCTCCTCTTTAATGAATTCTGTGTG 3′ (SEQ ID NO:14)

EcoRI enzyme restriction site is underlined

SalI enzyme restriction site is written in italic letters

The Ribosome Binding Site (RBS) is written in bold letters

The cloned PME gene from Erwinia chrysanthemi was excised from thePCR2.1 TOPO vector at the SalI and Notl sites, located in the primersused to PCR clone the genes. The SalI-Notl gene fragments were reclonedinto the pATP1 expression vector, pATP1E.chr.PME (FIG. 3).

Transformation of pATP1E.chr.PME vector into M15/pREP4 competent cells.

The pATP1E.chr.PME vector was transformed into competent M15/pREP4 cellsas described by the manufacturer (Qiagen).

Colonies containing the pATP1E.chr.PME vector were selected and used forinduction of expression of the Erwinia chrysanthemi PME gene.

Growth of E.coli

E.coli transformed with pATP1EchrPME vector was grown in LB-medium+100μg/ml ampicillin and 25 μg/ml kanamycin over night at 37° C. and 20 mlpre-culture was added to 800 ml LB-medium 100 μg/ml ampicillin and 25μg/ml kanamycin and incubated at 37° C. In total 3×800 ml was prepared.The cells were grown to the absorption at 600 nm was 0.7. 800 μl 1M IPTGwas added and after 4 hrs incubation at 37° C. the cells were harvested.

Preparation of Cell Free Extract

The cells were harvested by centrifugation at 10000 rpm for 10 min andresuspended in 50 ml extraction buffer (50 mM MES pH 6.8). The cellswere disrupted by sonication for 4×3 min with duty cycle of 70%. Inbetween and during the sonication treatment the sample was stored onice. The PME fraction (the supernatant) was obtained aftercentrifugation at 10000 rpm for 10 mm.

Chromatography

The PME was purified according to the following procedure. Alloperations were performed at 4° C. The supernatant obtained as describedabove was separated by cation exchange chromatography. A 50 ml samplewas applied to a CM-Sepharose™CL-6B (50 ml column material) and washedwith buffer A: 50 mM MES pH 6.8. The majority of the proteins did notbind to the column but the PME was absorbed and after washing off theunbound proteins with buffer A the bound proteins were eluted with anincreasing NaCl gradient from 0-1 M NaCl in total 450 ml. The flow was0.5 ml/min and fractions of 2.5 ml were collected. The proteinabsorption profile was measured at 280 nm.

All fractions were analysed for PME activity and protein. The proteincontent was measured spectrophotometrically with the BioRad method.

The fractions containing PME activity were pooled and used as enzymeextract for enzymatic PME treatment of pectin. SDS-PAGE revealed thatthis partially purified E. chrysanthemi PME fraction only contained 3-4proteins.

In order to purify the PME to homogeneity 1 ml of the pooled fractionwas concentrated by dialysis using Centricon filter system. Bufferexchange to 50 mM Tris, 0.1M NaCl pH 7 was done on the same system. 200μl concentrated sample was then applied to a Sephacryl™S-200 (2.6×70 cm)gel filtration column. The column was equilibrated with 50 mM Tris, 0.1M NaCl pH 7. The flow was 0.5 ml/min and fractions of 0.5 ml werecollected.

The fractions containing PME activity were pooled and concentrated usingCentricon as described above. The concentrated sample was then appliedto a Superdex™ G-75 which was equilibrated with 50 ml Tris, 0.1 M NaClpH 7. The now was 0.5 ml/min and fractions of 2 ml were collected. Thefractions containing PME activity were pooled and concentrated.

Enzyme Activity

PME catalyses the cleavage of methylester groups from pectin. During thepurification steps PME was detected by a fast method using methyl redindicator test. Due to cleavage of methyl groups from galacturonicresidues in the pectin chain, carboxyl groups were formed and the pHdrops in the assay. The pH indicator—methyl red—changes colour at pHdrop from yellow (pH 6.2) to pink (pH 4.2). The assay contained 1 ml0.5% lime pectin (DE 70%) solubilized in 0.15 M NaCl pH 7 and 25 μlsample. The samples which showed positive methyl red test after 10 minincubation at 30° C. were then further measured by the titration method.(Versteeg et al. (1978) Lebensmittel.-Wiss. u. Technol., 11: 267-274)

With the titration method the assay contained 10 ml 0.5% lime pectinsolubilized in 0.15 M NaCl pH 6.8 and 10-100 μl sample. Titration wasperformed with 0.02 M NaOH and the reaction was measured at roomtemperature. An automatic titrator was used. (Versteeo et al. (1978)Lebensmittel.-Wiss. u. Technol., 11: 267-274)

SDS-PAGE/Western Blotting

The purity of the PME fraction was investigated by SDS-PAGE as discussedabove.

Antibody Production

Antibodies are raised against the enzyme of the present invention byinjecting rabbits with the purified enzyme and isolating theimmunoglobulins from antiserum according to procedures describedaccording to N Harboe and A Ingild (“Immunization, Isolation ofImmunoglobulins, Estimation of Antibody Titre” In A Manual ofQuantitative Immunoelectrophoresis, Methods and Applications, N HAxelsen, et al (eds.), Universitetsforlaget, Oslo, 1973) and by T GCooper (“The Tools of Biochemistry”, John Wiley & Sons. New York, 1977).

STUDY EXPERIMENT 1

During purification of PME the cell free extract was applied to a cationexchange column (CM-Sepharose CL-6B). PME binds strongly to a cationexchange column material at pH 6.8 whereas most of the proteins do notbind to the column and so elute in the wash volume. With increasing NaClgradient PME eluted into one peak (fraction 56-64).

After concentration the PME fraction was further purified using gelfiltration chromatogtaphy (Sephacryl S-200 column) and followed bySuperdex G-75 gel filtration. Fractions containing the highest PMEactivity were pooled. The enzyme activity was 65 U/ml.

The fraction was tested for pectin degrading activity by viscositydetermination with 1% pectin at pH 4.8. The results showed that after 24hrs no change in the viscosity was found.

SDS-PAGE showed only one protein band in the purified PME fraction witha MW of 36,000 D. Isoelectric focusing of PME showed that the pI was >9.

Characterization and Kinetic Data

Characterization of PME and optima determinations were all done with thetitration method as described in Materials and Methods.

pH optimum of PME activity was measured with 0.5% lime pectin in 0.15 MNaCl. The optimum was found around pH 7. The enzyme has pH optimum atneutral pH but at pH 5 70% of the maximal activity was still measured.

Temperature optimum was found at 48° C.

The temperature stability of PME was determined by incubating the enzymesample in Eppendorf tubes at various temperatures for 15 min. Afterincubation the enzyme activity was measured by traditional assay bytitric method. The stability of the enzyme activity was between 20°-50°C. Incubation for 15 min at 60° C. resulted in inactivation of theenzyme.

The affinity for lime pectin was determined by Hanes plot of differentpectin concentration versus activity. The k_(m) was calculated from thecurye to be 0.44 mg/ml. The km was determined with pectin with DE 70%and the results showed that the km value was in the same range as foundfor orange PME but was 10 times lower than found for fungal PME fromAspergillus. This means that the catalytic activity of the plant andbacteria enzymes are 10-fold higher than that of the fungal enzyme.

E. chrysanthemi PME could also de-esterify sugar beet pectin. The PMEactivity was measured as described in Materials and Methods except that1% sugar beet pectin solubilized in 0.15 M NaCl was used in the assay.

We have also found that the enzyme does not necessarily require NaCl foractivity. However, the activity is increased with addition of NaCl up to0.1-0.15 M NaCl. Higher concentrations of NaCl decreases the activity.

STUDY EXPERIMENT 2 Pectin Treated with E. chrysanthemi has SimilarProperties to Pectin Treated with Plant PME

Calcium sensitive pectin

The URS pectin was treated with E. chrysanthemi PME and the obtainedmodified pectin was characterized with respect to % DE and Calciumsensitivity (CS).

% DE CS GRINSTED ™ URS Pectin 81.1% 1.1 Pectin 2084-124-1 73.7% 2.2Pectin 2084-124-2 70.6% 14.5 Pectin 2084-125-2 66.6% gel. Pectin2084-125-1 62.8% gel.

The two pectins (Pectin 2084-125-2 and Pectin 2084-125-1) gelled withthe added calcium in the test because of the very, high Ca-sensitivityand it was therefore not possible to obtain a CS value. This very highCa-sensitivity is only obtainable with block-wise de-methylated pectins.

Enzymatic fingerprinting of the E. chrysanthemi modified pectins byusing pectin lyase or polygalacturonase showed that the E. chrysanthemiPME de-methylates pectin blockwise producing non polygalacturonic acidlike blocks (random blocks).

Effect of E. Chrysanthemi PME treated pectin on the viscosity andstability of protein drinks

SP in acidified milk % DE drink Viscosity at cP 2143-17.1 78.9% 0.15%10.2 2143-27 77.5% 0.15% 10.6

SP=The minimum concentration at which the pectin stabilize the acidifiedmilk drink.

De-esterification of the URS pectin from 81% to 78-79% changed theCa-sensitivity.

The enzymatic modified pectins were tested in the acidified milk drinkat the dosage concentrations of 0.1%, 0.15%, 0.175%, 0.2% and 0.25%. Thequality of the individual acidified milk drink produced was investigatedby the parameters such as whey separation, the sedimentation % and theviscosity.

The results showed that the enzymatic modified pectins stabilize theprotein at the low pectin concentration of 0.15%. Each of the sampleshad a low viscosity. The E. chrysanthemi PME modified pectins have lowerviscosity at the pectin concentration of 0.15% than the standardcommercial stabilizer.

By treating the mother URS pectin with E. chrysanthemi PME an unsuitablepectin was made suitable as a stabilizing agent in the acidified milkdrink and this treated pectin behave just as well as an excellentcommercial stabilizer.

METHODS Enzymatic Treatment of Pectin with PME from E. chrysanthemi

A batch of enzymated pectin was prepared as follows: 45 g pectin wasdissolved in hot water under efficient stirring. 15 g NaCl was added andthe volume adjusted to 1.8 l with water. This solution was stirred untilthe salt had dissolved. The pectin solution was cooled to 40° C. and thepH was increased to pH 6.5, using 1 N NaOH and efficient stirring. Anappropriate sample of E. chrysanthemi PME was added and the enzymaticreaction continued until the desired degree of esterification wasachieved. The pH was kept constant at pH 6.5 by automatic dosage of 1 NNaOH during the incubation period, and the enzymatic reaction wasfollowed by the consumption of NaOH.

When the pectin sample had reached the desired degree of esterificationthe NaOH addition was stopped, the pH of the solution lowered to about3.0 by addition of 2% HCl. The pectin solution was then heated to 70° C.for 5 min to completely inactivate the enzyme. The treated pectin wasprecipitated with 1 volume of isopropanol, washed with 60% isopropanoland pressed to about 50% dry matter. The treated pectin batch was thenair dried at 40° C. and finally milled to a dry powder.

Determination of Pectin Samples Calcium Sensitivity Index (CS)

Calcium sensitivity was measured by the protocol presented above.

Determination of Pectin Samples Degree of Esterification (%DE)

The degree of esterification was measured by the protocol presentedabove.

Acidified Milk Drink Test

Standardised skimmed milk (17% MSNF), prepared from mixing powdered milkwith. adequate volume of de-ionised water at 68° C. for 20 minutes andcooled down to 30° C., was acidified at 30° C. with 3.3%glucone-delta-lactone (GDL) to about pH 4. The pectin sample was addedas a sugar solution and stirred for 30 min. The acidified milk drink washomogenized at 200 bar at room temperature and then filled into sterile250 ml plastic bottles. It was then heated on a water bath at 75° C. for10 min with internals of shaking for 5 minutes. Finally, the drink wascooled to room temperature and then stored overnight at 5° C.

Viscosity Determination of the Acidified Milk Drink

The viscosity of a sample of the milk drink was determined using aBrookfield Viscometer 11T at rpm 30. After stirring for 30 sec theviscosity is measured.

Protein Stability Measured by a Centrifugation Test

40 g of a sample (e.g. acidified milk drink) is centrifugated at roomtemperature at 2300×g for 20 min. The supernatant is discarded and thecentrifuge glass placed upside down for 30 min. The glass is weighed andthe sedimentation % calculated as follows:${\% \quad {Sedimentation}} = {\frac{{{Wgt}\quad {of}\quad {glass}\quad {after}\quad {centri}} - {{Wgt}\quad {of}\quad {glass}}}{{Wgt}\quad {of}\quad {sample}} \times 100}$

Where Wgt=weight and centri=centrifugation

Construction of Bacillus Expression Plasmid pCS.

The Bacillus/E. coli shuttle vector pDP66K (obtained from Dr L.Dijkhuizen, Rijks. University of Groningen, the Netherlands), wasmodified to make it suitable for cloning and expression of differentgenes. Details on the pDP66K vector are described in Penninga D et al(1996). (It is to be noted that any other suitable shuttle vector—suchas a commercially available shuttle vector—could also be used in thisexperiment.) The pCS plasmid was generated by modifying the promoter,the cyclodextrin glycosyl transferase (cgt) signal sequence, deletingthe rest of the cyclodextrin glycosyl transferase gene and replacing thetranscription terminal sequence. The p32 promoter and cyclodextringlycosyl transferase signal sequence was modified by PCR to contain aNcoI site at the ATG codon in the 3′ end of the signal sequence. Thetranscription terminator sequence was replaced by a PCR amplifiedtranscription terminator sequence from the pUB110 plasmid (McKenzie, T.,Hoshino, T., Tanaka, T. and Sueoka, N. (1986) The nucleotide sequence ofpUB110: some salient features in relation to replication and itsregulation. Plasmid 15 (2), 93-103, and McKenzie, T., Hoshino, T.,Tanaka, T. and Sueoka, N. (1987) Correction. A revision of thenucleotide sequence and functional map of pUB110. Plasmid 17 (1),83-85). A 5′ BamHI and a 3′ HindIII enzyme restriction sites wereintroduced into the PCR amplified transcription terminator sequence forcloning purposes. The resultant pCS plasmid is shown in FIG. 5.

Generation of the PME Gene Fragments for Cloning into the Bacillus pCSPlasmid.

Two PME gene PCR products were amplified, one containing the full lengthcoding region were the second amino acid was changed from a leucine to avaline in order to introduce a Ncol site at the 5′ ATG start codon ofthe gene for cloning purposes. In the 3′ end a BamHI site was introducedfor cloning purposes. The second PME gene PCR product was amplifiedwithout the genes signal sequence, starting from the alanine amino acidcodon at position 25 at the amino acid level. The PCR product wasdesigned to contain a methionine in front of the starting alanine aminoacid in order to introduce a Ncol, necessary for cloning purposes. Inthe 3′ end of the PCR product a BamHI site was introduced. PCRamplification of the two PME sequences were verified by DNA sequencing.The two amplified PME sequences were cloned into the pHM plasmid usingthe Ncol and BamHI restriction enzymes while the PME sequence withoutthe signal sequence also was cloned into the pCS plasmid using the samerestriction enzymes.

Expression of the PME Gene in Bacillus.

The four different constructs were transformed into Bacillus substilisand cultured in 2 xYT media containing 50 mg/ml kanamycin.

Expression of the E. chrysanthemi PME Gene in Aspergillus niger.

Construction of the Expression Vector pPR42-FS-PMEA

For expression in Aspergillus niger the E. chrysanthemi PME gene wasfurnished with a fungal signal sequence derived from the Aspergillusniger PME gene (Khanh, N. Q. et al (1991) Gene 1, 71-77) comprising thefollowing sequence:

Aspergillus niger PME signal sequence:

ATGGTTAAGTCAATTCTTGCATCCGTTCTCTTTGCGGCGACTGCGCTGGCC (SEQ ID NO:15)

MetValLysSerIleLeuAlaSerValLeuPheAlaAlaThrAlaLeuAla (SEQ ID NO:16)

Three overlapping oligo nucleotide 5′ end primers P1, P2 and P3 wereused together with a 3′ end primer P4 to fuse the fungal signal sequenceto the 5′ of the coding sequence of the E. chrysanthemi PME gene by PCR.

P1: 5′-GCGGCGACTGCGCTGGCCATGTTAAAAACGATCTCTGGAACCC (SEQ ID NO:17)

P2: 5′-AAGTCAATTCGCATCCGTTCTCMGCGGCGACTGCGC (SEQ ID NO:18)

P3: 5′-TGAATTCTCATGGTTAAGCAAUCTTGCATCCG (SEQ ID NO:19)

P4: 5′-TACTAGTGTCAGGGTAATGTCGGC (SEQ ID NO:20)

The 5′ end primer P1 contains the 5′ end of the E. chrysanthemi PMEcoding sequence, underlined. The 5′ end primer P3 contains an EcoRIrestriction enzyme site, underlined, and the 3′ end primer P4 contains aSpeI restriction enzyme site, underlined, to facilitate cloning.

PCR was performed with the Expand High Fidelity PCR system (BoehringerMannheim) according to the instructions of the manufacturer.

The amplified DNA fragment containing the fungal signal sequence fusedto the E. chrysanthemi PME gene was cloned into the PCR 2.1-TOPO cloningvector (Invitrogen) according to the instructions of the manufacturer.The cloned DNA fragment was sequenced using a Thermo Sequenasefluorescent cycle sequencing kit (Amersham) and an ALF DNA sequencer(Pharmacia) following the recommendations of the manufacturer.

An EcoRI DNA fragment containing the fungal signal sequence and the E.chrysanthemi PME gene was excised from the PCR 2.1-TOPO cloning vector,and introduced into the fungal expression vector pPR42 (See WO 9838321A) containing the XlnB promoter and the TrpC terminator generating thevector pPR42-FS-PMEA (FIG. 4).

Transformation of Aspergillus niger.

The pPR42-FS-PMEA vector was transformed into an uridine auxotrophicmutant of Aspergillus niger using a protocol adapted from Van Someren etal (1991) Curr. Genet. 20, 293-299, using cotransformation with a A.niger orotidine-5′-phosphate-decarboxylase gene and selection forcomplementation of the uridine auxotrophic mutation (Goosen et al (1987)Curr. Genet. 11, 499-503).

For the purification of Aspergillus niger protoplasts spores from a PDA(Potato Dextrose Agar-Difco Lab. Detroit) plate, containing 5 mMuridine, incubated for 3-4 days at 30° C. are washed off in 10 ml ST (8g/l NaCl, 0.5 g/l Tween 20). One million spores pr ml are inoculated in200 ml growth medium in a 500 ml shaking flask.

The growth medium contains: 6 g/l NaNO₃, 1.5 g/l KH₂PO₄, 0.5 g/l MgSO₄,7H₂O, 0.5 g/l KCL, 10 mM (NH₄)₂SO₄, 0.2% Casing enzymatic hydrolysate(Sigma C-0626), 2% glucose, 0.5% Yeast extract (Difco 0127-17-9), 10 mMUridine, 10 mg/l EDTA, 4.4 mg/l ZnSO₄, 7H₂O, 1 mg/l MnCl₂, 4H₂O, 0.32mg/l CoCl₂, 6H₂O, 0.32 mg/l CuSO₄, 5H₂O, 0.22 mg/l (NH₄)₆Mo₇O₂₄, 4H₂O,1.47 mg/l CaCl₂, 2H₂O, 1.0 mg/l FeSO₄, 7H₂O, pH 6.0.

The flask is shaken at 230-250 rpm for 16-18 hours at 30° C. Themycelium is harvested using Miracloth and washed 2-3 times with SMC(1.33 M Sorbitol, 50 mM CaCl₂, 20 mM MES, pH 5.8). 1 g wet mycelium isresuspended in 20 ml of SMC containing 150 mg Lyzing enzyme (SigmaL-2265), in a sterile flask and incubated at 37° C., 80-100 rpm for 1-3hours until protoplasts are released. The protoplasts are harvested bypassing the suspension through 5 ml sterile glasswool followed bycentrifugation at 3000 rpm for 10 min. The protoplasts are washed twicewith 5-10 ml of STC (1.33 M Sorbitol, 50 mM CaCl₂, 10 mM Tris/HCl, pH7.5) and finally resuspended in STC at 1×10⁸ protoplasts/ml.

For the transformation, 0.2 ml of protoplasts are carefully mixed with5-10 mg DNA (pPr42-FS-PMEA), 0.5 mg Selection DNA (pGW635—containing theorotidine-5′-phosphate-decarboxylase gene(Goosen et al (1987) Curr.Genet. 11, 499-503)) and 50 ml PEG solution (0.25 g/ml PEG 6000, 50 mMCaCl₂, 10 mM Tris/HCl, pH 7.5). The mixture is incubated for 20 min. atroom temperature after which 2 ml of PEG solution is carefully added andmixed. After further incubation for 5 min at room temperature 6 ml ofSTC is added, and the mixture is carefully shaken. The mixture iscentrifuged at 300 rpm for 10 min, and most of the supernatant isremoved.

The protoplasts are resuspended in the remaining 1-2 ml of supernatant,mixed with TR soft agar (6 g/l NaNO₃, 1.5 g/l KH₂PO₄, 0.5 g/l MgSO₄,7H₂O, 0.5 g/l KCL, 1.22 M Sorbitol, 10 mg/l EDTA, 4.4 mg/l ZnSO₄, 7H₂O,1 mg/l MnCl₂, 4H₂O, 0.32 mg/l CoCl₂, 6H₂O, 0.32 mg/l CuSO₄, 5H₂O, 0.22mg/l (NH₄)₆Mo₇O₂₄, 4H₂O, 1.47 mg/l CaCl₂, 2H₂O, 1.0 mg/l FeSO₄, 7H₂O,0.8% agar (Difco 0140-01), pH 6.0, and plated on TR plates (As TR softagar but with 1.2% agar). Transformed colonies are picked and replicatedon fresh TR plates.

Growth of A. niger Transformants for Expression of the E. chrysanthemiPME Gene.

For the expression of the E. chrysanthemi PME gene in A. nigertransformants, spores (aprox 500,000 pr ml) are inoculated in inductionmedium (6 g/l NaNO₃, 1.5 g/l KH₂PO₄, 0.5 g/l MgSO₄, 7H₂O, 0.5 g/l KCL,10 mM NH₄)₂SO₄, 10 mg/l EDTA, 4.4 mg/l ZnSO₄, 7H₂O, 1 mg/l MnCl₂, 4H₂O,0.32 mg/l CoCl₂, 6H₂O, 0.32 mg/l CuSO₄, 5H₂O, 0.22 mg/l (NH₄)₆Mo₇O₂₄,4H₂O, 1.47 mg/l CaCl₂, 2H₂O, 1.0 mg/l FeSO₄, 7H₂O, 29.4 g/lNa₃citrate,2H₂O, 0.2% Casein enzymatic hydrolysate (Sigma C-0626) and 2%Xylose) and incubated at 30° C. with shaking. After 2-3 days samples ofthe supernatant are taken daily and analysed for PME activity.

STUDY EXPERIMENT 3

The PME expressed in Aspergillus niger was tested for activity by themethod described earlier. The specific activity was 600 U/mg protein.

Characterisation of the protein by SDS-PAGE revealed a protein with MWof 36 kDa. The protein was not glycosylated as indicated by the correctMW of the mature protein. MALDI TOF MS analysis of the recombinant PMEprotein also showed that the enzyme was not glycosylated. From the aminoacid sequence two potential N-glycosylation sites were detected.

The protein was N-terminal sequenced and the N-terminal sequence was: AT T Y N A V V. This result shows that both the fungal signal peptide andthe signal peptide from Erwinia are processed correctly and the matureprotein is produced without any N-terminal signal peptides.

The pH optimum of the E. chrysanthemi expressed in A. niger is pH 5.5 to7. The same pH profile was also seen for the E. chrysanthemi PMEexpressed in E. coli.

The Aspergillus expressed E. chrysanthemi PME was concentrated byultrafiltration using YM 10 membrane (Amicon) prior to enzymaticmodification of pectin.

Modification of Pectin with Aspergillus Expressed PME Obtainable fromErwinia chrysanthemi

Methods:

Enzymatic treatment of pectin with PME from E. chrysanthemi.

40 g GRINDSTED™ Pectin URS 1400 was dissolved in hot water underefficient stirring. 7.8 g NaCl was added and the volume adjusted to 1.33l with water. The pectin solution was stirred until all material haddissolved and then cooled to 37° C. pH was increased to 5.5, using 1 NNaOH and efficient stirring. 26 U of PME was added and pH andtemperature was kept constant at 5.5 and 37° C. by automatic dosage of1N NaOH during about two hours, until 8 ml 1 N NaOH was consumed. pH ofthe solution was lowered to 3.0 by addition of 2 N HCl to stop theenzymatic reaction. The pectin solution was then heated to 70° C. for 5min to completely inactivate the enzyme. The treated pectin wasprecipitated with one volume isopropyl alcohol, washed with 60 vol. %isopropyl alcohol and pressed to 50% dry matter. The treated pectin wasthen air dried at 50° C. and finally milled to a dry powder. 38.7 genzyme treated pectin was isolated.

Results:

The pectin treated with PME from E. chrysanthemi which was expressed inAspergillus was analysed with respect to % DE and calcium sensitivity(CS). The results showed that the enzymatic treated pectin has increasedcalcium sensitivity compared to the mother pectin. The enzymatictreatment results in a HE-pectin with significant higher calciumsensitivity. These are the same results which were obtained with the E.chrysanthemi PME expressed in E. coli.

% DE CS GRlNDSTED ™ Pectin URS 1400 82.1% 1.11 Enzymatic treated sample75.3% 2.01

Discussion

The bacterial PME (Erwinia chrysanthemi) has been isolated andcharacterized. URS pectin with DE 81% has been de-esterified with thisPME to modified pectins with varying DE%. The characterization of thepectin revealed that the pectin is a moderately Ca-sensitive pectin or avery Ca-sensitive pectin at a high DE%. We believe that this is thefirst time that a bacterial PME has been shown to de-methylate pectin ina block-wise manner.

With the specific embodiment of the present invention, the modifiedpectin with DE of 78% has been tested in a acidified milk drink system.The results showed that the modified pectin stabilises the protein inthe acidified milk drink at a concentration of 0.15% pectin. The URSpectin do only stabilise the proteins in the acidified milk drink testat a very high concentration of pectin (>0.5).

Likewise, we believe that this is the first time it has been shown thata bacterial PME can de-methylate pectin in a blockwise manner and, inaddition, produce a Ca-sensitive pectin and, in addition, wherein themodified pectin can stabilise acidified milk drink.

PROTOCOLS PROTOCOL I CALCIUM SENSITIVITY INDEX (CS)

Calcium sensitivity is measured as the viscosity of a pectin dissolvedin a solution with 57.6 mg calcium/g pectin divided by the viscosity ofexactly the same amount of pectin in solution, but without addedcalcium. A calcium insensitive pectin has a CS value of 1.

4.2 g pectin sample is dissolved in 550 ml hot water with efficientstirring. The solution is cooled to about 20° C. and the pH adjusted to1.5 with 1N HCl. The pectin solution is adjusted to 700 ml with waterand stirred. 145 g of this solution is measured individually into 4viscosity glasses. 10 ml water is added to two of the glasses (doubledeterminations) and 10 ml of a 250 mM CaCl₂ solution is added to theother two glasses under stirring.

50 ml of an acetate buffer (0.5 M, pH about 4.6) is added to all fourviscosity glasses under efficient magnetic stirring, thereby bringingthe pH of the pectin solution up over pH 4.0. The magnets are removedand the glasses left overnight at 20° C. The viscosities are measuredthe next day with a Brookfield viscometer. The calcium sensitivity indexis calculated as follows:${CS} = \frac{{Viscosity}\quad {of}\quad a\quad {solution}\quad {with}\quad 57.6\quad {mg}\quad {Ca}^{2 +}\text{/}g\quad {pectin}}{{Viscosity}\quad {of}\quad a\quad {solution}\quad {with}\quad 0.0\quad {mg}\quad {Ca}^{2 +}\text{/}g\quad {pectin}}$

PROTOCOL II DEGREE OF ESTERIFICATION (%DE)

To 50 ml of a 60% isopropanol and a 5% HCl solution is added 2.5 gpectin sample and stirred for 10 min. The pectin solution is filteredthrough a glass filter and washed with 15 ml 60% isopropanol/5% HClsolution 6 times followed by further washes with 60% isopropanol untilthe filtrate is free of chlorides. The filtrate is dried overnight at80° C.

20.0 ml 0.5 N NaOH and 20.0 ml 0.5 N HCl is combined in a conical flaskand 2 drops of phenolphtalein is added. This is titrated with 0.1 N NaOHuntil a permanent colour change is obtained. The 0.5 N HCl should beslightly stronger than the 0.5N NaOH. The added volume of 0.1 N NaOH isnoted as V₀.

0.5 g of the dried pectin sample (the filtrate) is measured into aconical flask and the sample is moistened with 96% ethanol. 100 ml ofrecently boiled and cooled destilled water is added and the resultingsolution stirred until the pectin is completely dissolved. Then 5 dropsof phenolphtalein are added and the solution titrated with 0.1 N NaOH(until a change in colour and pH is 8.5). The amount of 0.1 N NaOH usedhere is noted as V₁. 20.0 ml of 0.5 N NaOH is added and the flask shakenvigously, and then allowed to stand for 15 min. 20.0 ml of 0.5 N HCl isadded and the flask is shaken until the pink colour disappears. 3 dropsof phenolphtalein are then added and then the resultant solution istitrated with 0.1 N NaOH. The volume 0.1 N NaOH used is noted as V₂.

The degree of esterification (% DE: % of total carboxy groups) iscalculated as follows:${\% \quad {DE}} = \frac{V_{2} - V_{0}}{V_{1} + \left( {V_{2} - V_{0}} \right)}$

PROTOCOL III DRINK TEST 1. Introduction

The following describes a protocol that only uses about 1.7 g pectin toas little as possible. The methods used to evaluate the performance ofthe system are viscometry, centrifugal sedimentation, and particle sizedetermination.

2. Materials and Methods

2.1 Materials

Skim milk powder with approx. 36% protein was obtained from MejeriernesFælles Indkøb (Kolding, Denmark). Pectins for testing were obtained bytreatment of a pectin with a modified PME according to the presentinvention. These pectins may have different properties such as degree ofesterification and molecular weight, depending on the type of modifiedPME used.

2.2 Preparation of Milk Drink

The milk drinks were made by mixing an acidified milk solution and apectin solution. followed by further processing.

A milk solution was made by dissolving 17% (w/w) skimmilk powder indistilled water at 68° C. and stirring for 30 min. The milk solution wasthen acidified to pH 4.1 at 30° C. by addition of 3% (w/w)glucono-d-lactone (GDL).

The pectin solution was made up in several steps. First pectin was drymixed with dextrose at a 3:2 weight ratio, and then a 1.11% (w/w)solution of this mixture in distilled water was made. The last step inthe preparation of the pectin solution was to add sucrose to an endconcentration of 17.8% (w/w).

Milk drinks were then prepared by mixing 1 part of milk solution with1.13 parts (w/w) of pectin solution, followed by heat treatment (seesection 3.2) and homogenisation at 20-22 MPa and 20° C. using a Mini JetHomogeniser (Burgaud et. al. 1990). By following this procedure, thefinal concentration of pectin in the milk drink was 0.3% (w/w). Allsamples were produced in duplicate stored at 5° C. and tested forviscosity, particle size and sedimentation the following day.

2.3 Viscosity Measurement

The viscosity was measured using a Bohlin VOR Rheometer system (BohlinInstruments, Metric Group Ltd., Gloucestershire, Great Britain).Thermostatation was achieved by a Bohlin lower-plate temperature controlunit. The viscosity was measured at a shear rate of 91.9 s⁻¹. Themeasuring temperature was 20° C., and the samples were held at 20° C.for approximately 1 hour before measurement. The measuring system usedwas C 14 (a coaxial cylindrical system). The torque element used was0.25 g cm. Integration time was 5 s, measurement internal was 30 s, andno autozero was used. Instrumental control and primary data processingwere done on a PC with the Bohlin Rheometer Software version 4.05.

2.4 Particle Size Measurement

The particle mean diameter, D[4.3], was measured with a MalvernMastersizer Micro Plus (Malvern Instruments Limited, Worcestershire,UK). Instrumental settings were: presentation code: 5NBD, and AnalysisModel: polydisperse. Instrumental control and primary data processingwere done on a PC with Mastersizer Microplus for Windows, version 2.15.

Ultrafiltration permeate obtained from a batch of acidified milk drinkmade with pectin no. 4 was used for dilution. Ultrafiltration was doneusing a DDS UF Lab 20-0.36 module fitted with GR61PP membranes, having amolecular weight cut-off of 20.000 Da.

2.5 Sedimentation

Sedimentation measurements were performed by centrifugation of thesamples using an IEC Centra-8R Centrifuge (International Equipment,Needham Hts, Mass. USA). 2.5 g acidified milk drink was centrifuged for25 min at 20° C. and 2400 g. The supernatant was removed, the tubes wereleft up side down for 15 min, and the weight of the sediment wasdetermined and expressed as a percentage (of the amount of milk drinkused). Duplicate measurements were made of each sample.

3. Results and Discussion

3.1 Size of Test System

This new system is small compared to the previous test systems but itstill maintains the same properties as the existing test system based on550 g acidified milk drink. The easiest way to make a model system fortesting pectins in acidified milk drinks would be to simply mix stirredacidified milk drink with a pectin solution, and make the measurementson this mixture. This also has the advantage that it can be donevirtually at any scale. However, Glahn and Rolin showed that ahomogenisation reduces the amount of pectin needed for stabilisation andthat both homogenisation and heat treatment have very considerableeffects on stability. Since both homogenisation and heat treatment wereincluded in the existing system at 550 g scale, as they are inindustrial processes, both treatments also needed to be present in thesmall scale system.

In industry both upstream (before heating) and downstream (afterheating) homogenisation is used. In this model system we chose to putthe homogenisation in after heat treatment because this gives a morehomogeneous sample and thereby makes it easier to obtain reproduciblemeasurements of e.g. viscosity.

To achieve a reproducible homogenisation with the Mini Jet Homogeniser,and to compensate for various losses during sample transfer, it wasdesirable to operate with 40 ml of sample at the homogenisation stage.Since only 8-9 ml was needed for the tests (2.5 ml for viscometry, 5 mlfor sedimentation, and 0.5-1 ml for particle size determination), thestep that required the largest amount of sample was the homogenisation,and the result was therefore that the existing test system was scaleddown from 550 g to 40 g milk drink.

3.2 Heat Treatment

To make the scaled down system mimic the existing test system as closelyas possible it was desirable to make modifications to the heat treatmentstep. With the existing 550 g system heating took place in a 600 mlBlue-cap bottle for 30 min in a 75° C. water bath, with stirring every 5minutes.

With the new 40 g system the heat treatment was done in a 50 ml plasticcentrifuge tube placed inside a 600 ml Blue-cap bottle filled withwater. Here 75° C. in the water bath gave too strong a heating, probablybecause the thermal conductivity of water is larger than that ofcoagulated milk. Different temperatures between 70 and 75° C. weretherefore tested, and it was found that 72° C. for 30 minutes, withoutstirring, gave a good approximation to the temperature profile in thelarge system.

3.3 Testing of Small Scale System

If a milk drink stabilised with a pectin treated with a modified PMEaccording to the present invention showed little sedimentation and smallparticles, then that indicates a good pectin to use and moreover isindicative that the modified PME according to the present invention issuitable for such a use.

4. Conclusion

A system for testing the stabilising power of pectins in acidified milkdrinks has successfully been scaled down from 550 g to 40 g milk drink,meaning that the required amount of pectin is reduced from ca. 1.7 g toca. 0.15 g. This is small enough to allow screening of experimentalpectin samples treated with modified pectins according to the presentinvention. A high correlation between results obtained for particlesize, viscosity and sedimentation between the two methods has beendemonstrated. The scaled down method is relatively simple, although itstill contains both heating and homogenisation which is consideredimportant for industrial relevance.

PROTOCOL IV PME ACTIVITY

PME catalyses the cleavage of methylester groups from pectin. During thepurification steps PME can be detected by a fast method using methyl redindicator test. Due to cleavage of methyl groups from galacturonic acidresidues in the pectin chain, carboxyl groups are formed and the pH willthen drop in the assay. The pH indicator—methyl red—changes colour at pHdrop from yellow (pH 6.2) to pink (pH 4.2). Typically, the assay willcontain 1 ml 0.5% Grindsted™ Pectin 1450 (DE 70%) (supplied by DaniscoIngredients, Danisco A/S) solubilized in 0.15 M NaCl pH 7 and 25 μlsample. The samples which then show positive methyl red test after 10min incubation at 30° C. are then further measured by the titrationmethod (Versteeg et al (1978) Lebensmittel.-Wiss. u. Technol., 11:267-274).

With the titration method the assay will typically contain 10 ml 0.5%lime pectin (Grindsted™ Pectin 1450—supplied by Danisco Ingredients,Danisco A/S) solubilized in 0.15 M NaCl pH 6.8 and 10-100 μl sample.Titration is performed with 0.02 M NaOH and the reaction is measured atroom temperature. An automatic titrator can be used (Versteeg et al.(1978) Lebensmittel.-Wiss. u. Technol., 11: 267-274).

For convenience we now present a Table indicating the codes used for theamino acids.

THREE LETTER AMINO ACID ABBREVIATION ONE LETTER SYMBOL Alanine Ala AArginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

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21 1 1101 DNA Erwinia chrysanthemi 1 atgttaaaaa cgatctctgg aaccctcgcgctgtcgctga ttatcgctgc cagcgtacat 60 caggcacagg cagcgaccac ctacaacgctgtggtatcaa aatcctccag cgacggcaaa 120 acaatcaaaa ctattgccga cgcaattgccagcgccccag caggcagcac gccgttcgtc 180 attttgatca agaacggcgt ctataatgaacgcctgacga ttacccgcaa taacctgcat 240 ctgaaaggcg aaagtcgtaa cggtgcggtcattgcggctg ccacggcggc gggcaccctg 300 aaatcggacg gcagcaagtg gggaacggcaggcagcagca ccatcaccat cagcgccaag 360 gatttcagcg cccagtcgct gaccattcgcaacgactttg atttcccggc caatcaggcc 420 aaaagcgaca gcgacagcag taaaatcaaggacacgcagg cagttgcgct ctatgtcacc 480 aaaagcggcg accgcgccta cttcaaagacgtcagtctgg tcggctatca ggacacgctg 540 tatgtttccg gcggccgcag tttcttctccgactgccgta tcagcggcac ggttgacttt 600 atctttggcg acggcaccgc gctgttcaacaactgcgatc tggtttcccg ctatcgcgct 660 gatgtgaaaa gcggcaatgt ttccggctacctgaccgcgc ccagcaccaa catcaatcag 720 aagtatggcc tggtgatcac caacagtcgcgtgatacggg aaagtgactc tgtaccggcg 780 aaaagctacg ggctgggtcg cccctggcatccaacaacaa ccttctctga tggccgttac 840 gcgaatccga acgctattgg tcagaccgttttcctgaaca ccagcatgga taatcatatt 900 tatggttggg acaagatgtc cggcaaggacaaaaacggca acaccatctg gttcaacccg 960 gaagattccc gtttcttcga gtacaaatcctatggcgcgg gagcggcggt gagcaaagac 1020 cgccgacagt tgactgacgc acaggcggcagagtacacgc agagcaaagt cctgggcgac 1080 tggacgccga cattaccctg a 1101 2 366PRT Erwinia chrysanthemi 2 Met Leu Lys Thr Ile Ser Gly Thr Leu Ala LeuSer Leu Ile Ile Ala 1 5 10 15 Ala Ser Val His Gln Ala Gln Ala Ala ThrThr Tyr Asn Ala Val Val 20 25 30 Ser Lys Ser Ser Ser Asp Gly Lys Thr IleLys Thr Ile Ala Asp Ala 35 40 45 Ile Ala Ser Ala Pro Ala Gly Ser Thr ProPhe Val Ile Leu Ile Lys 50 55 60 Asn Gly Val Tyr Asn Glu Arg Leu Thr IleThr Arg Asn Asn Leu His 65 70 75 80 Leu Lys Gly Glu Ser Arg Asn Gly AlaVal Ile Ala Ala Ala Thr Ala 85 90 95 Ala Gly Thr Leu Lys Ser Asp Gly SerLys Trp Gly Thr Ala Gly Ser 100 105 110 Ser Thr Ile Thr Ile Ser Ala LysAsp Phe Ser Ala Gln Ser Leu Thr 115 120 125 Ile Arg Asn Asp Phe Asp PhePro Ala Asn Gln Ala Lys Ser Asp Ser 130 135 140 Asp Ser Ser Lys Ile LysAsp Thr Gln Ala Val Ala Leu Tyr Val Thr 145 150 155 160 Lys Ser Gly AspArg Ala Tyr Phe Lys Asp Val Ser Leu Val Gly Tyr 165 170 175 Gln Asp ThrLeu Tyr Val Ser Gly Gly Arg Ser Phe Phe Ser Asp Cys 180 185 190 Arg IleSer Gly Thr Val Asp Phe Ile Phe Gly Asp Gly Thr Ala Leu 195 200 205 PheAsn Asn Cys Asp Leu Val Ser Arg Tyr Arg Ala Asp Val Lys Ser 210 215 220Gly Asn Val Ser Gly Tyr Leu Thr Ala Pro Ser Thr Asn Ile Asn Gln 225 230235 240 Lys Tyr Gly Leu Val Ile Thr Asn Ser Arg Val Ile Arg Glu Ser Asp245 250 255 Ser Val Pro Ala Lys Ser Tyr Gly Leu Gly Arg Pro Trp His ProThr 260 265 270 Thr Thr Phe Ser Asp Gly Arg Tyr Ala Asn Pro Asn Ala IleGly Gln 275 280 285 Thr Val Phe Leu Asn Thr Ser Met Asp Asn His Ile TyrGly Trp Asp 290 295 300 Lys Met Ser Gly Lys Asp Lys Asn Gly Asn Thr IleTrp Phe Asn Pro 305 310 315 320 Glu Asp Ser Arg Phe Phe Glu Tyr Lys SerTyr Gly Ala Gly Ala Ala 325 330 335 Val Ser Lys Asp Arg Arg Gln Leu ThrAsp Ala Gln Ala Ala Glu Tyr 340 345 350 Thr Gln Ser Lys Val Leu Gly AspTrp Thr Pro Thr Leu Pro 355 360 365 3 22 PRT Artificial Sequence SITE(1) Preferably Xaa is Ala, Val, Gly or Thr 3 Xaa Xaa Xaa Gln Xaa Xaa XaaXaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 204 31 DNA Artificial Sequence Description of Artificial Sequence Primer 4agtcgacgtg tatgttaaaa acgatctctg g 31 5 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 5 agcggccgca attcgtcagggtaatgtcgg 30 6 16 DNA Artificial Sequence Description of ArtificialSequence Primer 6 gtaaacgacg gccagt 16 7 19 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 ggaaacagct atgaccatg 19 8 17DNA Artificial Sequence Description of Artificial Sequence Primer 8gattatccat gctggtg 17 9 18 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 cggcgtctat aatgaacg 18 10 16 DNA ArtificialSequence Description of Artificial Sequence Primer 10 gcgacagcga cagcag16 11 19 DNA Artificial Sequence Description of Artificial SequencePrimer 11 ccgtggcagc cgcaatgac 19 12 56 DNA Artificial SequenceDescription of Artificial Sequence pSPORT1 EcoR1 - HindIII fragment 12aagcttggat cctctagagc ggccgccgac tagtgagctc gtcgacccgg gaattc 56 13 47DNA Artificial Sequence Description of Artificial Sequence Primer 13cacacagaat tcattaaaga ggagaaatta acccgtcgac ccgggag 47 14 47 DNAArtificial Sequence Description of Artificial Sequence Primer 14ctcccgggtc gacgggttaa tttctcctct ttaatgaatt ctgtgtg 47 15 51 DNAAspergillus niger 15 atggttaagt caattcttgc atccgttctc tttgcggcgactgcgctggc c 51 16 17 PRT Aspergillus niger 16 Met Val Lys Ser Ile LeuAla Ser Val Leu Phe Ala Ala Thr Ala Leu 1 5 10 15 Ala 17 43 DNAArtificial Sequence Description of Artificial Sequence Primer 17gcggcgactg cgctggccat gttaaaaacg atctctggaa ccc 43 18 40 DNA ArtificialSequence Description of Artificial Sequence Primer 18 aagtcaattcttgcatccgt tctctttgcg gcgactgcgc 40 19 34 DNA Artificial SequenceDescription of Artificial Sequence Primer 19 tgaattctca tggttaagtcaattcttgca tccg 34 20 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 20 tactagtgtc agggtaatgt cggc 24 21 8 PRTErwinia chrysanthemi 21 Ala Thr Thr Tyr Asn Ala Val Val 1 5

What is claimed is:
 1. A process for treating a pectin with pectin methyl esterase (PME) to produce a PME-treated high ester pectin, comprising the step of contacting a pectin with an Erwinia PME, having a pH optimum with lime pectin of about pH 7.0, that is capable of block-wise de-esterification of the pectin, to produce a PME-treated high ester pectin that contains from about 70% to about 80% ester groups.
 2. The process according to claim 1 wherein the PME has a molecular weight of about 36,000 D and/or a pI of about>9 and/or a temperature optimum with lime pectin of about 48° C.
 3. The process according to claim 1 wherein the pME comprises the amino acid sequence shown as SEQ.I.D. No.2, or an amino acid sequence having PME activity with at least 75% homology to SEQ ID No:2.
 4. The process according to claim 1 wherein the PME has the amino acid sequence shown as SEQ.I.D. No.2, or an amino acid sequence having PME activity with at least 75% homology to SEQ ID No:2.
 5. The process according to claim 1 wherein the PME has the amino acid sequence shown as SEQ.I.D. No.2.
 6. The process according to claim 1 wherein the PME has been expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No.1, or a nucleotide sequence with at least 75% homology to SEQ ID No:1, or combinations thereof.
 7. The process according to claim 1 wherein the PME has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No.1, or a nucleotide sequence with at least 75% homology to SEQ ID No:1.
 8. The process according to claim 1 wherein the PME has been expressed by a nucleotide sequence having the nucleotide sequence shown as SEQ.I.D. No.1.
 9. The process according to claim 1 wherein the PME has been prepared by use of known recombinant DNA techniques.
 10. The process according to claim 1 wherein the pectin is in contact with the PME in the presence of sodium ions.
 11. The process according to claim 10 wherein the sodium ions are derived from NaCl, NaNO₃, or Na₂SO₄ or combinations thereof.
 12. The process according to claim 1 wherein the process includes the further step of isolating the PME-treated pectin from remaining active PME.
 13. The process according to claim 12 wherein the PME treated pectin contains from about 72% to about 80% ester groups.
 14. The process according to claim 12 wherein the PME treated pectin contains from about 74% to about 80% ester groups.
 15. The process according to claim 12 wherein the PME treated pectin contains from about 76% to about 80% ester groups.
 16. The process according to claim 12 wherein the PME treated pectin contains from about 77% to about 80% ester groups.
 17. The process according to claim 1 wherein the process includes the further step of adding the PME-treated pectin to a medium that is suitable for human or animal consumption.
 18. The process according to claim 17 wherein the medium is an aqueous solution.
 19. The process according to claim 18 wherein the aqueous solution is a beverage.
 20. The process according to claim 17 wherein the medium is an acidic environment.
 21. The process according to claim 20, wherein the acidic environment has a pH of from about 3.5 to about 5.5.
 22. The process according to claim 21 wherein the acidic environment has a pH of about
 4. 23. The process according to claim 19, wherein the beverage is a acidified milk drink.
 24. The process according to claim 17 herein the medium of comprises a protein.
 25. The process according to claim 22 wherein the protein is derived from or is obtainable from or is in a dairy product.
 26. The process according to claim 22 wherein the protein is derived from or is obtainable from or is in a plant product.
 27. The process according to claim 20, wherein the acidic environment has a pH of from 4 to about 5.5.
 28. A method for reducing the number of ester groups in a pectin in a block-wise manner, comprising the step of contacting a pectin with a pectin methyl esterase (PME) to produce a PME-treated pectin that contains about 70% to about 80% ester groups and which PME comprises the amino acid sequence shown as SEQ.I.D. No.2 or an amino acid sequence with at least 75% homology to SEQ ID No:2, wherein said PME is not a plant PME and is capable of block-wise de-esterification of the pectin.
 29. A method for de-esterifying two or more adjacent galacturonic acid residues of a pectin on a pectin chain, comprising the step of contacting a pectin with a pectin methyl esterase (PME) to produce a PME-treated pectin that contains about 70% to about 80% ester groups and which PME comprises the amino acid sequence shown as SEQ.I.D. No.2 or an amino acid sequence with at least 75% homology to SEQ ID No:2, wherein said PME is not a plant PME and is capable of block-wise de-esterification of the pectin.
 30. The process according to claim 28 or 29 wherein the PME is obtained from a micro-organism.
 31. The process according to claim 30 wherein the PME is obtained from a bacterium.
 32. The process according to claim 17, wherein the medium is enriched with a protein.
 33. The process according to claim 32 wherein the protein is derived from or is obtainable from or is in a dairy product.
 34. The process according to claim 32 wherein the protein is derived from or is obtainable from or is in a plant product. 